Note: Descriptions are shown in the official language in which they were submitted.
WO 2023/012584
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1
ENGINEERED TCR COMPLEX AND METHODS OF USING SAME
RELATED APPLICATION/S
This application claims the benefit of priority of European Patent Application
No. 21189516.4 filed on August 3,2021, and US Provisional Patent Application
No. 63/345,966
filed on May 26. 2022, the contents of which are incorporated herein by
reference in their entirety.
SEQUENCE LISTING STATEMENT
The file entitled 9325 Lxml, created on 21 July 2022, comprising 274,432
bytes, submitted
concurrently with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to engineered TCR
complex
and methods of using same.
Cancer immunotherapy, including cell-based therapy, antibody therapy and
cytokine
therapy, has emerged in the last couple of years as a promising strategy for
treating various types
of cancer owing to its potential to evade genetic and cellular mechanisms of
drug resistance and
to target tumor cells while sparing healthy tissues.
Cell-based therapy using e.g. T cells having a T cell receptor (TCR) specific
for an antigen
differentially expressed in association with an MHC class I molecule on cancer
cells or having a
chimeric antigen receptors (CAR) comprising an antigen recognition moiety
[e.g., a single chain
variable fragment (scFv)] and a T-cell activation moiety were shown to exert
anti-tumor effects in
several types of cancers, e.g. hematologic malignancies. However, TCRs are
limited in their
recognition spectrum and the MHC class and in addition, when introducing an
exogenous TCR to
a T cell there is a risk of hybridization between exogenous and endogenous
chains, which may
induce recognition of autoantigens [Van Loenen MM, et al. Proc Natl Acad Sci U
S A.
2010;107:10972-7]. CART cells on the other hand, despite their advantages,
have critical flaws
that need to be solved to allow for full utilization of the technology in
clinical treatments, including
e.g. severe side effects, vigorous expansion in the presence of heavy tumor
burden leading to tumor
I ysi s syndrome and cytokine release syndrome, development of tumor escape
variants which have
lost the target antigen during treatment [Morgan RA et al. (2010) Mol Ther.
18: 843-51; Brudno
JN et al. (2016) Blood. American Society of Hematology; page 3321-30; and
Grupp SA et al.
(2013) N Engl J Med 368: 1509-181. Further, using allogeneic T cells for
therapy imposes the
risk of harmful recognition of autoantigens leading to graft versus host
disease (GVHD).
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Antibody-based cancer immunotherapies, such as monoclonal antibodies, antibody-
fusion
proteins, and antibody drug conjugates (ADCs) depend on recognition of cell
surface molecules
that are differentially expressed on cancer cells relative to non-cancerous
cells and/or immune-
checkpoint blockade. Binding of an antibody-based immunotherapy to a cancer
cell can lead to
cancer cell death via various mechanisms, e.g., antibody-dependent cell-
mediated cytotoxicity
(ADCC), complement-dependent cytotoxicity (CDC), direct cytotoxic activity of
the payload from
an antibody-drug conjugate (ADC) or suppressive checkpoint blockade. Many of
these
mechanisms initiate through the binding of the Fc domain of cell-bound
antibodies to specialized
cell surface receptors (Fc receptors) on hematopoietic cells. In recent years
another type of
antibody-based therapy was suggested, namely anti-CD3 bi-specific antibodies,
such as
Mosunetuzumab, Odronextamab and Blinatumomab. These hi-specific antibodies
engage CD3
positive T cells with tumor-associated antigen and thus promote T cells to
attack tumor cells.
Immunotherapies combining principles of antibody-based therapy, CAR T cells
and/or
TCR based immunotherapy have been disclosed [see e.g. Choi BD et al. (2019)
Nature Publishing
Group; 37: 1049-58; Chandran and Klebanoff (2019) Immunological Reviews.
290:127-147;
Benjamin R. (2020) Lancet 396: 1885-94; Liu et al. (2021) Sci. Trans'. Med.
13, eabb5191; Helsen
C.W et al. (2018) Nature Communications 9:3049; Baeuerle P.A. et al. (2019)
Nature
Communications 10:2087_1.
Additional background art includes:
International Patent Application Publication Nos. W02019222275, W02021035170
and
W02015143224;
US Patent Application Publication Nos. US20190388472 and US20190070248;
Japanese Patent No. JP2017514471; and
Russian Patent No. RU2725542.
SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is
provided a
T cell receptor (TCR) complex comprising a TCR comprising a TCR alpha
polypeptide and a TCR
beta polypeptide, wherein the TCR is devoid of an antigen-binding domain, and
a CD3 polypeptide
devoid of a heterologous antigen-binding domain; and wherein the TCR complex
is capable of
being presented on a surface of a T cell expressing same.
According to an aspect of some embodiments of the present invention there is
provided a
T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR
beta
polypeptide, wherein the TCR is devoid of an antigen-binding domain, and
wherein the TCR alpha
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and beta polypeptides comprise amino acid modifications enabling presentation
of the TCR as a
TCR complex on a surface of a T cell expressing same.
According to some embodiments of the invention, the TCR comprises a
heterologous
dimerizing moiety.
According to some embodiments of the invention, the heterologous dimerizing
moiety
comprises a cysteine residue at each of the TCR alpha polypeptide and the TCR
beta polypeptide.
According to some embodiments of the invention, the TCR alpha polypeptide and
the TCR
beta polypeptide comprise a human TCR alpha polypeptide and a human TCR beta
polypeptide.
According to some embodiments of the invention, the TCR alpha polypeptide and
the TCR
beta polypeptide comprise a chimeric human and murine TCR alpha polypeptide
and a chimeric
human and murine TCR beta polypeptide.
According to an aspect of some embodiments of the present invention there is
provided a
T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR
beta
polypeptide, wherein the TCR is devoid of an antigen-binding domain and a
heterologous
extracellular binding domain, and wherein the TCR alpha and beta polypeptides
comprise amino
acid modifications enabling presentation of the TCR as a TCR complex on a
surface of a T cell
expressing same, the modifications comprise:
(i) a T48C amino acid substitution corresponding to the human TCR alpha
polypeptide
as set forth in SEQ ID NO: 9, and a S57C amino acid substitution corresponding
to the human TCR
beta polypeptide as set forth in SEQ ID NO: 12; and/or
(ii) the TCR alpha polypeptide and the TCR beta polypeptide comprise a
chimeric
human and murine TCR alpha polypeptide and a chimeric human and murine TCR
beta
polypeptide.
According to an aspect of some embodiments of the present invention there is
provided a
T cell receptor (TCR) comprising a human TCR alpha polypeptide and a human TCR
beta
polypeptide, wherein the TCR is devoid of an antigen-binding domain, and
wherein the TCR alpha
and beta polypeptides comprise amino acid modifications enabling presentation
of the TCR as a
TCR complex on a surface of a T cell expressing same, the modifications
comprise:
(i) T48C, S116L, G119V and F120L amino acid substitutions corresponding to
the
human TCR alpha polypeptide as set forth in SEQ ID NO: 9, and a 557C mutation
corresponding
to the human TCR beta polypeptide as set forth in SEQ ID NO: 12; and/or
(ii) P91S, E92D, 593V and 594P amino acid substitutions corresponding to
the human
TCR alpha polypeptide as set forth in SEQ ID NO: 9, and E18K, S22A, F1331,
E/V136A and
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Q139H amino acid substitutions corresponding to the human TCR beta polypeptide
as set forth in
SEQ ID NO: 12.
According to some embodiments of the invention, the modifications of (ii)
further comprise
S116L, G119V and F120L amino acid substitutions corresponding to the human TCR
alpha
polypeptide as set forth in SEQ ID NO: 9.
According to some embodiments of the invention, the modifications further
comprise
Si 16L, G1 19V and Fl 20L amino acid substitutions corresponding to the human
TCR alpha
polypeptide as set forth in SEQ ID NO: 9.
According to some embodiments of the invention, the modifications of (ii)
comprise P9 1S,
E92D, S93V and S94P amino acid substitutions corresponding to the human TCR
alpha
polypeptide as set forth in SEQ ID NO: 9, and E18K, 522A, F1331, E/V136A and
Q139H amino
acid substitutions corresponding to the human TCR beta polypeptide as set
forth in SEQ ID NO:
12.
According to some embodiments of the invention, the TCR alpha polypeptide
comprises
an amino acid sequence selected from the group consisting of SEQ ID NO: 14.
17, 20-23, 47, 111,
114 and 117-120.
According to some embodiments of the invention, the TCR alpha polypeptide
comprises
an amino acid sequence selected form the group consisting of SEQ ID NO: 14, 20-
23, 47, 111 and
117-120.
According to some embodiments of the invention, the TCR alpha polypeptide
comprises
an amino acid sequence selected form the group consisting of SEQ ID NO: 8, 14,
17 and 20-23.
According to some embodiments of the invention, the TCR alpha polypeptide
comprises
an amino acid sequence selected form the group consisting of SEQ ID NO: 14, 17
and 20-23.
According to some embodiments of the invention, the TCR alpha polypeptide
comprises
an amino acid sequence selected form the group consisting of SEQ ID NO: 14 and
20-23.
According to some embodiments of the invention, the TCR beta polypeptide
comprises an
amino acid sequence selected from the group consisting of SEQ ID NO: 15-16, 18-
19, 24-25, 112-
113, 115-116 and 121-122.
According to some embodiments of the invention, the TCR beta polypeptide
comprises an
amino acid sequence selected form the group consisting of SEQ ID NO: 10-11,15-
16, 18-19 and
24-25.
According to some embodiments of the invention, the TCR beta polypeptide
comprises an
amino acid sequence selected form the group consisting of SEQ ID NO: 15-16, 18-
19 and 24-25.
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According to an aspect of some embodiments of the present invention there is
provided at
least one polynucleotide encoding the TCR.
According to an aspect of some embodiments of the present invention there is
provided a
transduced cell expressing the TCR or the at least one polynucleotide.
5
According to an aspect of some embodiments of the present invention there
is provided a
transduccd T cell expressing the TCR complex or TCR or the at least one
polynucleotide.
According to an aspect of some embodiments of the present invention there is
provided a
method of producing a TCR expressing cell, the method comprising introducing
into a cell the at
least one polynucleotide, under conditions which allow expression of the TCR.
According to an aspect of some embodiments of the present invention there is
provided a
method of expressing a TCR in a T cell, the method comprising introducing into
a T cell the at
least one polynucleotide, under conditions which allow expression of the TCR.
According to some embodiments of the invention, the introducing is effected in-
vitro or ex-
vivo.
According to some embodiments of the invention, the cell does not express an
endogenous
TCR.
According to some embodiments of the invention, the T cell does not express an
endogenous TCR.
According to some embodiments of the invention, the method further comprising
dowm-egulating expression of the endogenous TCR.
According In some embodiments of the invention, the method further comprising
downregulating expression of the endogenous TCR prior to the introducing.
According to an aspect of some embodiments of the present invention there is
provided a
method of treating a disease associated with a pathological cell in a subject
in need thereof, the
method comprising administering to the subject a therapeutically effective
amount of the T cell;
and a therapeutic composition capable of binding the pathological cell and the
TCR complex,
thereby treating the disease in the subject.
According to an aspect of some embodiments of the present invention there is
provided the
T cell; and a therapeutic composition capable of binding a pathological cell
and the TCR complex,
for use in treating a disease associated with the pathological cell in a
subject in need thereof.
According to an aspect of some embodiments of the present invention there is
provided a
method of treating a disease associated with a pathological cell in a subject
in need thereof, the
method comprising administering to the subject a therapeutically effective
amount of the T cell;
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and a therapeutic composition capable of binding the pathological cell and a
TCR complex
comprising the TCR, thereby treating the disease in the subject.
According to an aspect of some embodiments of the present invention there is
provided the
T cell; and a therapeutic composition capable of binding a pathological cell
and a TCR complex
comprising the TCR, for use in treating a disease associated with the
pathological cell in a subject
in need thereof.
According to some embodiments of the invention, the T cell is allogeneic to
the subject.
According to an aspect of some embodiments of the present invention there is
provided an
article of manufacture comprising a packaging material packaging the cell; and
a therapeutic
composition capable of binding a pathological cell and a TCR complex
comprising the TCR.
According to an aspect of some embodiments of the present invention there is
provided an
article of manufacture comprising a packaging material packaging the T cell;
and a therapeutic
composition capable of binding a pathological cell and the TCR complex.
According to some embodiments of the invention, the therapeutic composition
comprises
an anti-CD3 antibody.
Non-limiting examples of anti-CD3 antibodies that can be used with specific
embodiments
of the invention include L2K, TR66, OKT3, SP34, UCHT1, F6A, humanized UCHTL
SK7 and
H1T3A.
According to some embodiments of the invention, the anti-CD3 antibody is
selected from
the group consisting of L2K, TR66 and OKT3.
According to some embodiments of the invention, the pathological cell
expresses CD19
and the therapeutic composition comprises Blinatumomab.
According to some embodiments of the invention, the pathological cell
expresses EpCAM
and the therapeutic composition comprises MT110.
According to some embodiments of the invention, the pathological cell is a
cancerous cell.
According to some embodiments of the invention, the cancer is selected from
the group
consisting of lymphoma, leukemia, glioblastoma, colon cancer, gastric cancer,
pancreatic cancer,
ovarian cancer, lung cancer and skin cancer.
Unless otherwise defined, all technical and/or scientific terms used herein
have the same
meaning as commonly understood by one of ordinary skill in the art to which
the invention pertains.
Although methods and materials similar or equivalent to those described herein
can be used in the
practice or testing of embodiments of the invention, exemplary methods and/or
materials are
described below. In case of conflict, the patent specification, including
definitions, will control. In
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addition, the materials, methods, and examples are illustrative only and are
not intended to be
necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example
only, with
reference to the accompanying drawings. With specific reference now to the
drawings in detail, it
is stressed that the particulars shown are by way of example and for purposes
of illustrative
discussion of embodiments of the invention. In this regard, the description
taken with the drawings
makes apparent to those skilled in the art how embodiments of the invention
may be practiced.
In the drawings:
FIGs. 1A-B shows schematic representations of the engineered TCR complex of
some
embodiments of the invention. Figure lA demonstrates a TCR complex comprising
TCR alpha
and beta chains that are devoid of the variable alpha and beta regions. This
TCR is referred to
herein as "blunt truncated TCR (BluT)". Figure 1B demonstrates a TCR complex
comprising
mismatch pairing of exogenous TCR alpha and beta chains devoid of variable
regions with
endogenous TCR alpha and beta chains.
FIGs. 2A-B show schematic representations of the engineered TCR of some
embodiments
of the invention. When indicated, 3 mutations were introduced in the
transmembrane domain of
the alpha chain: S116L, G119V and F120L (marked as LVL). When indicated,
cysteines were
introduced in both the alpha and beta chains (marked as CYS): T48C in the
alpha chain and S57C
in the beta chain. When indicated, several mutations were introduced in the
extracellular domain
of the alpha and beta chains, as follows: alpha chain mutations P91S, E92D,
593V, 594P; beta
chain mutations: E18K, S22A, F1331, E/V136A, Q139H (marked as mm). When
indicated,
cysteines were introduced in both the alpha and beta chains: L12C in the alpha
chain and S17C in
the beta chain, Y43C in the alpha chain and L63C in the beta chain, S61C in
the alpha chain and
R79C in the beta chain, L12C in the alpha chain and F14C in the beta chain,
V22C in the alpha
chain and F14C in the beta chain. YlOC in the alpha chain and Sl7C in the beta
chain. T45C in the
alpha chain and D59C in the beta chain, L50C in the alpha chain and 557C in
the beta chain, 561C
in the alpha chain and S57C in the beta chain, T45C in the alpha chain and
S77C in the beta chain,
S15C in the alpha chain and V13C in the beta chain, or S15C in the alpha chain
and E15C in the
beta chain. When indicated, several mutations were introduced in the alpha and
beta chains, as
follows: alpha chain mutations S21F, T32I, A72T and beta chain mutations E18K,
H23R, D39P,
S54D (marked as Des). F5P2A - Furin-V5 tag sequence combined with P2A.
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FIGs. 3A-B demonstrate re-expression of CD3 in endogenous TCR negative T cells
following expression of BluT. Figure 3A shows generation of CD3-/TCR- T cells
by
electroporation with Cas9 RNP and gRNA targeting the TCR alpha chain (SEQ ID
NO: 1) followed
by magnetic beads purification of the CD3-/TCR- T cells. Figure 3B shows re-
expression of CD3
in the T cells shown in Figure 3A following infection with a construct
encoding a truncated alpha
chain comprising an additional cysteine and several transmembrane hydrophobic
mutations and a
truncated beta chain comprising an additional cysteine, (TRAC(Cys,LVL)-P2A-
TRBC1(Cys),
SEQ ID NO: 2 as compared to no expression following infection with a construct
encoding only a
truncated alpha chain (TRAC, SEQ ID NO: 3), as determined by flow cytometry
using an anti-
CD3 OKT3 antibody. EGFP serves as a marker of infection.
FIG. 4 demonstrates re-expression of CD3 in endogenous TCR negative T cells
following
expression of BluT comprising at least the T48C mutation in the alpha chain
and the S57C mutation
in the beta chain. Endogenous TCR negative T cells shown in Figure 3A were
infected with the
indicated constructs (see Figure 2B for detailed description of each
construct) and evaluated by
flow cytometry using an anti-CD3 OKT3 antibody. EGFP serves as a marker of
infection.
FIG. 5 demonstrates re-expression of CD3 in endogenous TCR negative T cells
generated
by targeting the endogenous beta or alpha and beta chains, following
expression of BluT
comprising the T48C and LVL mutations in the alpha chain and the S57C mutation
in the beta
chain. CD3-/TCR- T cells were generated by electroporation with Cas9 RNP and
gRNA targeting
the TCR beta chain (SEQ ID NO: 45), or both TCR alpha and beta chains (SEQ ID
NO: 1 and 45)
followed by magnetic beads purification of the CD3-/TCR- T cells. Following,
cells were infected
with the indicated constructs (see Figure 2B for detailed description of each
construct) and
evaluated by flow cytometry using an anti-CD3 OKT3 antibody. EGFP serves as a
marker of
infection.
FIG. 6 demonstrates that expression of BluT comprising mutations leading to an
additional
disulfide bond between the alpha and beta chains other than the T48C in the
alpha chain and the
557C in the beta chain do not enable CD3 re-expression. Endogenous TCR
negative T cells shown
in Figure 3A were infected with the indicated constructs (see Figure 2B for
detailed description of
each construct) and evaluated by flow cytometry using an anti-CD3 OKT3
antibody. EGFP serves
as a marker of infection.
FIG. 7 demonstrates re-expression of CD3 in endogenous TCR negative T cells
following
expression of BluT comprising minimal fnurine amino acid substitutions.
Endogenous TCR
negative T cells shown in Figure 3A were infected with the indicated
constructs (see Figure 2B for
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detailed description of each construct) and evaluated by flow cytometry using
an anti-CD3 OKT3
antibody. EGFP serves as a marker of infection.
FIG. 8A demonstrates the effect of removing 6-21 amino acids from the N-
terminal of the
alpha chain of BluT on re-expression of CD3 in endogenous TCR negative T
cells. Endogenous
TCR negative T cells shown in Figure 3A were infected with the indicated
constructs (see Figure
2B for detailed description of each construct) and evaluated by flow cytometry
using an anti-CD3
OKT3 antibody. EGFP serves as a marker of infection.
FIG. 8B demonstrates no expression of CD3 in endogenous TCR negative T cells
following
expression of constructs disclosed by International Patent Application
Publication NO.
W02020138371. Endogenous TCR negative T cells shown in Figure 3A were infected
with Ba9
(SEQ ID NO: 83/84) or ct6 (SEQ ID NO: 81/82) and evaluated by flow cytometry
using an anti-
CD3 OKT3 antibody. EGFP serves as a marker of infection.
FIG. 9 demonstrates that T cells expressing BluT in combination with a hi-
specific T cell
engager induce effective in-vitro tumor cell lysis. Endogenous TCR negative T
cells shown in
Figure 3A were infected with BluT comprising a T48C mutation and the LVL
mutations and a
truncated beta chain comprising a S57C mutation (SEQ ID NO: 125/126) were
incubated with
Raji-F.Luc CD19+ lymphoma cells at the indicated E: T ratios with or without
Blinatumomab (a
CD19 BiTE) and firefly luciferase activity was measured to determine
cytotoxicity. CD3 negative
anti-CD19 CAR-T cells (SEQ ID NO: 127/128) and un-modified CD3 positive TCR
positive T
cells were used as positive controls. Shown is percentage of relative lysis
calculated by dividing
tested group RLU (Relative Light Units) on the non treated only tumor group
RLU. Results are
presented in (%). ****p<0.0001, ***p<0.001 by two-way Anova test.
FIG. 10 demonstrates that T cells expressing BluT in combination with a bi-
specific T cell
engager induce effective in-vivo anti-cancer effect Endogenous TCR negative T
cells shown in
Figure 3A were infected with BluT comprising a T48C mutation and the LVL
mutations and a
truncated beta chain comprising a S57C mutation (SEQ ID NO: 125/126).
Following, mice were
transplanted with CD19+ Raji cells in combination the BluT transduced T cells
and, when
indicated, treated with the anti-CD19 BiTE Blinatumomab. CD3 negative anti-
CD19 CAR-T cells
(SEQ ID NO: 127/128) and un-modified CD3 positive TCR positive T cells were
used as positive
controls. Saline treatment served as a negative control. Shown is mice
survival as Kaplan-Meier
curves.
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DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to engineered TCR
and
methods of using same.
Before explaining at least one embodiment of the invention in detail, it is to
be understood
5 that the invention is not necessarily limited in its application to the
details set forth in the following
description or exemplified by the Examples. The invention is capable of other
embodiments or of
being practiced or carried out in various ways.
Cancer immunotherapy has emerged in the last couple of years as a promising
strategy for
treating various types of cancer. Cell-based therapy using e.g. T cells having
an engineered T cell
10 receptor (TCR) or CAR T cells, antibody-based cancer inimunotherapies, and
combinations
thereof were shown to exert anti-tumor effects in several types of cancers.
Whilst reducing specific embodiments of the present invention to practice, the
present
inventors designed and expressed a truncated TCR that is devoid of the
variable regions of the
TCR alpha and beta chains which was presented as part of a TCR complex on the
surface of T
cells devoid of an endogenous TCR (Example 1 of the Examples section which
follows). Further,
T cells expressing the truncated TCR had an anti-tumor effect both in-vitro
and in vivo only in the
presence of an anti-CD3 mediator (Examples 2-3 of the Examples section which
follows).
Consequently, specific embodiments of the present teachings suggest T cells
genetically
engineered to express the truncated TCR in combination with a therapeutic
composition capable of
binding a pathological cell on the one hand and the TCR complex on the other
hand (e.g. a T cell
engager antibody) to treat diseases associated with pathologic cells (e.g.
cancer).
Thus, according to an aspect of the present invention, there is provided a T
cell receptor
(TCR) complex comprising a TCR comprising a TCR alpha polypeptide and a TCR
beta
polypeptide, wherein said TCR is devoid of an antigen-binding domain, and a
CD3 polypeptide
devoid of a heterologous antigen-binding domain; and wherein said TCR complex
is capable of
being presented on a surface of a T cell expressing same.
According to an additional or an alternative aspect of the present invention,
there is
provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and
a human TCR
beta polypeptide, wherein said TCR is devoid of an antigen-binding domain, and
wherein said TCR
alpha and beta polypeptides comprise amino acid modifications enabling
presentation of said TCR
as a TCR complex on a surface of a T cell expressing same.
According to an additional or an alternative aspect of the present invention,
there is
provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and
a human TCR
beta polypeptide, wherein said TCR is devoid of an antigen-binding domain and
a heterologous
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extracellular binding domain, and wherein said TCR alpha and beta polypeptides
comprise amino
acid modifications enabling presentation of said TCR as a TCR complex on a
surface of a T cell
expressing same, said modifications comprise:
(i) a T48C amino acid substitution corresponding to the human TCR alpha
polypeptide
as set forth in SEQ ID NO: 9, and a S57C amino acid substitution corresponding
to the human TCR
beta polypeptide as set forth in SEQ ID NO: 12; and/or
(ii) said TCR alpha polypeptide and said TCR beta polypeptide comprise a
chimeric
human and murine TCR alpha polypeptide and a chimeric human and murine TCR
beta
polypeptide.
According to an additional or an alternative aspect of the present invention,
there is
provided a T cell receptor (TCR) comprising a human TCR alpha polypeptide and
a human TCR
beta polypeptide, wherein said TCR is devoid of an antigen-binding domain, and
wherein said TCR
alpha and beta polypeptides comprise amino acid modifications enabling
presentation of said TCR
as a TCR complex on a surface of a T cell expressing same, said modifications
comprise:
(i) T48C,
S116L, G119V and F120L amino acid substitutions corresponding to the
human TCR alpha polypeptide as set forth in SEQ ID NO: 9, and a S57C amino
acid substitution
corresponding to the human TCR beta polypeptide as set forth in SEQ ID NO: 12;
and/or
(ii)
P91S, E92D, S93V and S94P amino acid substitutions corresponding to
the human
TCR alpha polypeptide as set forth in SEQ ID NO: 9 and E18K, S22A, F1331,
E/V136A and
Q139H amino acid substitutions corresponding to the human TCR beta polypeptide
as set forth in
SEQ ID NO: 12.
As used herein, the term "T cell receptor (TCR)" refers to a receptor
comprising a hetero-
dimer of a TCR alpha polypeptide and a TCR beta polypeptide, which is capable
of being presented
(or presentable) as a TCR complex on a surface of a T cell expressing same.
As used herein, the term "TCR complex" refers to a complex formed by the
association of
CD3 with a TCR.
As used herein, the term "CD3" refers to the polypeptide expression product of
the CD3G,
CD3D, CD3E or CD247 gene (Gene ID 917, 915, 916, 919, respectively), and
includes CD3y,
CD3, CD3 E and CD3zeta.According to specific embodiments, CD3 is human CD3.
According to a specific embodiment, the CD3 is endogenous to the cell it is
expressed in.
According to a specific embodiment, the CD3 refers to the human CD3y
polypeptide, such
as provided in the following Accession No. NP_000064 or SEQ ID NO: 4.
According to a specific embodiment, the CD3 refers to the human CD36, such as
provided
in the following Accession Nos. NP_000723, NP_001035741 or SEQ ID NO: 5.
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According to a specific embodiment, the CD3 refers to the human CD3, such as
provided
in the following Accession No. NP_000724 or SEQ ID NO: 6.
According to a specific embodiment, the CD3zeta (CD247) refers to the human,
such as
provided in the following Accession No. NP 000725, NP 932170, NP 001365444,
NP_001365445 or SEQ ID NO: 7.
Herein, the -CD3 polypeptide" or -CD3 chain" refers to full length CD3 or a
fragment or
a homolog thereof, which comprises a signaling domain and maintains at least
the capability of
CD3 of being presented as a TCR complex on a surface of a T cell expressing
same. Such
homologues can be, for example, at least 70 %, at least 75 %, at least 80 %,
at least 81 %, at least
82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87
%, at least 88 %, at least
89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94
%, at least 95 %, at least
96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or
homologous to the
polypeptide set forth in SEQ ID NO: 4-7.
Thus, for example, a TCR complex can be composed of a CD3y chain, a CD3o
chain, two
CDR chains, a homodimer of CD3zeta chains, a TCR alpha polypeptide, and a TCR
beta
polypeptide.
Methods of determining presentation of a TCR complex are well known in the art
and
include for example Flow cytometry or immunostaining using an anti-CD3
antibody, or an anti-
TCR beta antibody.
The TCR disclosed herein is devoid of an antigen-binding domain.
According to specific embodiments, the CD3 polypeptide is devoid of a
heterologous
antigen-binding domain.
According to specific embodiments, wherein the TCR comprises a human TCR alpha
and
a human TCR beta polypeptides comprising the amino acid modifications enabling
presentation
of the TCR as a TCR complex on a surface of a T cell expressing same, the CD3
polypeptide may
comprise a heterologous antigen-binding domain.
As used herein, the term "heterologous" refers to an amino acid sequence or
residue which
is not native to the recited amino acid sequence (e.g. TCR alpha polypeptide,
a TCR beta
polypeptide, a CD3 polypeptide) at least in localization or is completely
absent from the native
sequence of the recited amino acid sequence.
Hence, according to specific embodiments, all the components of the TCR
complex are
devoid of an antigen-binding domain.
As used herein, the phrase "antigen-binding domain" refers to the domain that
contains an
amino acid sequence which confers binding to an antigen in a specific manner
i.e., Kd of 10-4M or
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lower, of a TCR or an antibody. Typically, such a domain in the context of the
present invention
includes the variable domains of the TCR alpha and beta chains of a TCR or the
variable domains
or the heavy and light chains of an antibody.
Thus, the TCR is truncated of a naturally occurring antigen-binding domain
i.e., is devoid
of a naturally occurring antigen-binding domain of the naturally occurring
TCR.
In other words, the TCR alpha and beta polypeptides; and the CD3 polypeptide
of some
embodiments of the invention, are not transl ati onal 1 y fused to an antigen-
binding domain
The TCR alpha and beta polypeptides or CD3 polypeptide of some embodiments may
be
attached to an antigen-binding domain not as a translational fusion (e.g. an
antibody comprising
an antigen-binding domain having a binding specificity to the TCR complex).
According to other specific embodiments, the TCR alpha and/or beta
polypeptides are not
attached to a non-translationally fused antigen-binding domain.
According to specific embodiments, the TCR and/or CD3 polypeptide is devoid of
a
heterologous extracellular binding domain capable of binding a target e.g.
that is presented on a
cell surface of a target cell of a T cell. In other words, the TCR alpha and
beta polypeptides and/or
CD3 polypeptide are not translationally fused to a heterologous extracellular
binding domain
capable of binding a target.
As used herein, the phrase "extracellular binding domain capable of binding a
target" refers
to a proteinaceous moiety having a binding affinity i.e., Kd of 10-4M or
lower, to a target of interest
(or binding pair), such as for example a target being presented on a target
cell. Non-limiting
examples of binding domains include the binding domain of a receptor, the
binding domain of a
ligand, the binding domain of a hormone (e.g. leptin), a tag and an antigen-
binding domain, as
further described hereinabove.
According to specific embodiments, the TCR is devoid of an antigen-binding
domain and
a heterologous extracellular binding domain.
In other words, according to specific embodiments, the extracellular domains
of the TCR
polypeptides consist of the constant domains or fragments or homologs thereof
or the constant and
hinge domains or fragments or homologs thereof of TCR alpha and beta chains.
Assays for testing binding are well known in the art and include, but not
limited to flow
cytometry, immunostaining, bio-layer interferometry Blitz assay, HPLC,
surface plasmon
resonance (e.g. Biacore).
Herein, the "TCR alpha polypeptide" refers to a fragment of a TCR alpha chain
or a
homolog thereof, comprising an extracellular domain devoid of an antigen-
binding domain (i.e.,
devoid of a variable domain), a transmembrane domain, and optionally an
intracellular domain,
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which maintains at least the capability of a TCR alpha chain of being
presented as a TCR complex
on a surface of a T cell expressing same.
As used herein, "TCR alpha" or "TCR alpha chain" refers to the polypeptide
expression
product of the TRA gene (Corresponding to human Gene ID 6955) or a homolog
thereof. According
to specific embodiments, TCR alpha refers to the human TCR alpha. According to
specific
embodiments, TCR alpha refers to the mouse TCR alpha.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence of a TCR alpha constant domain or a fragment or homolog thereof, a
TCR alpha hinge
region, a TCR alpha transmembrane domain, and a TCR alpha intracellular
domain.
According to specific embodiments, the TCR alpha polypeptide is less than 170,
less than
160, less than 155, or less than 152 amino acids long, each possibility
represents a separate
embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide is at least 120,
at least 125,
at least 130, at least 135, at least 140 or at least 150 amino acids long,
each possibility represents a
separate embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide is 135 ¨ 151
amino acids
lone.
According to specific embodiments, the TCR alpha polypeptide is about 135
amino acids
long.
According to specific embodiments, the TCR alpha polypeptide is 140 ¨ 151
amino acids
long.
According to specific embodiments, the TCR alpha polypeptide is about 141
amino acids
long.
According to specific embodiments, the TCR alpha polypeptide is about 151
amino acids
long.
According to specific embodiments, the TCR alpha polypeptide is about 145
amino acids
long.
According to specific embodiments, the TCR alpha polypeptide is about 150
amino acids
long.
According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
polypeptide is at least 84, at least 85, at least 86, at least 87, at least
88, at least 89, at least 90 amino
acids in length.
According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
polypeptide is at least 84 amino acids in length.
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According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
polypeptide is at least 88 amino acids in length.
According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
polypeptide is 94 amino acids in length.
5
According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
polypeptide is at least the sequence between amino acid 11 of SEQ ID NO: 9 to
amino acid 94 of
SEQ ID NO: 9, however homologous sequences and sequence modifications of SEQ
ID NO: 9 are
further contemplated as further described hereinabove and below.
According to specific embodiments, the TCR alpha constant domain of the TCR
alpha
10
polypeptide is at least the sequence between amino acid 7 of SEQ ID NO: 9
to amino acid 94 of
SEQ ID NO: 9, however homologous sequences and sequence modifications of SEQ
ID NO: 9 are
further contemplated as further described hereinabove and below.
According to specific embodiments, the TCR alpha peptide comprises the entire
constant
domain of TCR alpha.
15
According to specific embodiments, the TCR alpha polypeptide comprises a
murine TCR
alpha polypeptide.
A non-limiting example of a murine TCR alpha polypeptide that can be used with
specific
embodiments of the invention is provided in Uniprot ID A0A075B662 (SEQ ID NO:
8).
According to specific embodiments, the TCR alpha polypeptide comprises a human
TCR
alpha polypeptide.
A non-limiting example of a human TCR alpha polypeptide devoid of a variable
domain is
provided in Uniprot ID P01848 (SEQ ID NO: 9).
Herein the "TCR beta polypeptide" refers to a fragment of a TCR beta chain or
a homolog
thereof, comprising an extracellular domain devoid of an antigen-binding
domain (i.e., devoid of a
variable domain), a transmembrane domain, and optionally an intracellular
domain, which
maintains at least the capability of a TCR beta chain of being presented as a
TCR complex on a
surface of a T cell expressing same.
As used herein, "TCR beta" or "TCR beta chain" refers to the polypeptide
expression
product of the TRB gene (Corresponding to human Gene ID 6957) or a homolog
thereof and
includes TRBC1 (Corresponding to the human Gee ID 28639) and TRBC2
(Corresponding to
human Gene ID 28638) or homologs of same.
According to specific embodiments, TCR beta refers to TRBC1.
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According to specific embodiments, TCR beta refers to TRBC2.
According to specific embodiments, TCR beta refers to the human TCR beta.
According to specific embodiments, TCR beta refers to the mouse TCR beta.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence of a TCR beta constant domain or a fragment or homolog thereof. a TCR
beta hinge
region, a TCR beta transmembrane domain, and a TCR beta intracellular domain.
According to specific embodiments, the TCR beta polypeptide is less than 250,
less than
200, less than 190, or less than 188 amino acids long, each possibility
represents a separate
embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide is at least 140,
at least 150,
at least 160, at least 170, at least 180 or at least 185 amino acids long,
each possibility represents a
separate embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide is 170-187 amino
acids long.
According to specific embodiments, the TCR beta polypeptide is about 172 amino
acids
long.
According to specific embodiments, the TCR beta polypeptide is about 178 amino
acids
lone.
According to specific embodiments, the TCR beta polypeptide is about 183 amino
acids
long.
According to specific embodiments, the TCR beta polypeptide is about 187 amino
acids
long.
According to specific embodiments, the TCR beta constant domain of the TCR
beta
polypeptide is at least 100, at least 110, at least 120, at least 125 amino
acids in length.
According to specific embodiments, the TCR beta constant domain of the TCR
beta
polypeptide is 130 amino acids in length.
According to specific embodiments, the TCR beta peptide comprises the entire
constant
domain of TCR beta.
According to specific embodiments, the TCR beta polypeptide comprises a murine
TCR
beta polypeptide.
Non-limiting examples of murine TCR beta polypeptides that can be used with
specific
embodiments of the invention are provided in SEQ ID NOs: 10-11.
According to specific embodiments, the TCR beta polypeptide comprise a human
TCR beta
polypeptide.
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Non-limiting examples of human TCR beta polypeptides devoid of a variable
domain are
provided in SEQ ID NOs: 12-13.
In order to enable presentation of a TCR having a human TCR alpha and beta
polypeptides
devoid of a binding domain (an antigen-binding domain, a heterologous
extracellular binding
domain) the present inventors have envisaged modifications in the sequences of
the human TCR
alpha and beta polypeptides. Non-limiting examples of such modifications
include insertion of a
heterol ogous di meri zi ng moiety and insertion of a minimal murine amino
acid sequence.
Thus, according to specific embodiments, the TCR alpha polypeptide and/or TCR
beta
polypeptide comprise a chimeric human and murine TCR alpha polypeptide and/or
TCR beta
polypeptide. Thus, for Example the TCR alpha and/or beta polypeptide may be
based on human
TCR alpha and/or beta comprising a short (e.g. 3 ¨ 20) murine amino acid
sequence. Such
sequences are known in the art and disclosed in e.g. Sommermeyer D, et al. J
Immunol. 2010;
Bialer G, et al. J Immunol. 2010, the contents of which are fully incorporated
herein by reference.
Thus, according to specific embodiments, the TCR alpha polypeptide comprises a
human
TCR alpha polypeptide comprising the amino acid substitutions (or changes, or
mutations) P9 1S,
E92D, S93V and S94P corresponding to TCR alpha amino acid sequence as set
forth in SEQ ID
NO: 9.
According to specific embodiments, the TCR beta polypeptide comprises a human
TCR
beta polypeptide comprising the amino acid substitutions (or changes or
mutations) E18K, S22A,
F1331, E/V136A, Q139H corresponding to TCR beta amino acid sequence as set
forth in SEQ ID
NO: 12.
As used herein, the phrase "corresponding to SEQ ID NO: 9" intends to include
the
corresponding amino acid residue relative to any other TCR alpha amino acid
sequence.
As used herein, the phrase "corresponding to SEQ ID NO: 12" intends to include
the
corresponding amino acid residue relative to any other TCR beta amino acid
sequence.
Thus, according to specific embodiments, the TCR alpha polypeptide comprises
SEQ ID
NO: 14 and the TCR beta polypeptide comprises SEQ ID NO: 15 or 16.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
111 and the TCR beta polypeptide comprises SEQ ID NO: 112 or 113.
According to specific embodiments, the TCR alpha and/or beta polypeptide may
he based
on human TCR alpha and/or beta comprising the hinge region of mouse TCR alpha
and/or beta,
respectively.
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According to specific embodiments, the TCR alpha and/or beta polypeptide may
comprise
the transmembrane domain and optionally the intracellular domain of human TCR
alpha and/or
beta, respectively, and the extracellular domain of mouse TCR alpha and/or
beta, respectively.
According to specific embodiments, the TCR comprises a heterologous dimerizing
moiety.
According to specific embodiments, the heterologous dimerizing moiety is
located in the
extracellular domain of the TCR.
According to specific embodiments, the heterologous dimerizing moiety is
located in the
constant domain of the TCR.
According to specific embodiments, the heterologous dimerizing moiety is
located in the
hinge domain of the TCR.
As used herein, the term "dimerizing moiety" refers to a heterologous amino
acid sequence
capable of forming a TCR alpha polypeptide ¨ TCR beta polypeptide hetero-
dimer. Such an amino
acid may include for example an amino acid sequence comprising at least two
cysteine residues,
one in the TCR alpha polypeptide and the second in the TCR beta polypeptide,
enabling the
formation of a disulfide bond between the thiol groups. Another example is
dimerization domain
or an amino acid sequence enabling steric formation of a dimer. Such sequences
are known to the
skilled in the art. Methods of determining dimerization are known in the art,
including but not
limited to immunoprecipitation, size exclusion chromatography, fast protein
liquid
chromatography (FPLC), multi-angle light scattering (SEC-MALS) analysis, SDS-
PAGE
analysis, nano-DSF, yeast two-hybrid system (e.g. RRS) and flow cytometry.
Any known dimerizing moiety known in the art can be used with specific
embodiments of
the invention. Non-limiting examples of dimerizing moieties that can be used
with specific
embodiments of the invention include a heterologous cysteine residue at each
of the TCR alpha
and TCR beta polypeptides, an Fc domain of an antibody (not comprising the
antibody antigen-
binding domain), murine amino acid sequences capable of forming a heterodimer
such as the short
murine amino acid sequences described hereinabove, FRB-FKBP, leucine zipper.
According to specific embodiments, the TCR alpha and beta polypeptides each
comprise at
least one heterologous cysteine.
According to specific embodiments, the heterologous cysteine is located in the
extracellular
domain of each of the TCR alpha and beta polypeptides.
According to specific embodiments, the heterologous cysteine is located in the
hinge or
constant domain of each of the TCR alpha and beta polypeptides.
According to a specific embodiment, the TCR alpha polypeptide comprises a T48C
amino
acid substitution corresponding to the TCR alpha amino acid sequence as set
forth in SEQ ID NO:
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9 and the TCR beta polypeptide comprises a S57C amino acid substitution
corresponding to the
TCR beta amino acid sequence as set forth in SEQ ID NO: 12.
Thus, according to specific embodiments, the TCR alpha polypeptide comprises
SEQ ID
NO: 17 and the TCR beta polypeptide comprises SEQ ID NO: 18 or 19.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
114 and the TCR beta polypeptide comprises SEQ ID NO: 115 or 116.
As each of the TCR alpha and beta polypeptides comprise an endogenous cysteine
located
in the hinge regions of the polypeptides forming a single disulfide bond
between the alpha and beta
polypeptides, the addition of at least one heterologous cysteine results in
the presence of at least
two cysteine residues in each of the TCR alpha and beta polypeptides enabling
the formation of at
least two disulfide bonds between the TCR alpha and TCR beta polypeptides.
Thus, according to specific embodiments, the TCR alpha and beta polypeptides
each
comprise at least two cysteine residues capable of forming at least two
disulfide bonds between the
alpha and beta polypeptides.
According to specific embodiments, the at least two cysteine residues are
located in the
extracellular domain of each of the TCR alpha and beta polypeptides.
According to specific embodiments, at least one cysteine residue is located in
the hinge
region of each of the TCR alpha and beta polypeptides and at least one
cysteine residue is located
in the hinge or constant domain of each of the TCR alpha and beta
polypeptides.
The terms "TCR alpha polypeptide" and "TCR beta polypeptide" also encompass
functional homologues (naturally occurring or synthetically/recombinantly
produced), which
exhibit the desired activity (i.e., being presented as a TCR complex on a
surface of a T cell
expressing same). Such homologues can be, for example, at least 70 %, at least
75 %, at least 80
%, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %,
at least 86 %, at least 87
%, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %,
at least 93 %, at least 94
%, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %
or 100 % identical or
homologous to the polypeptide set forth in SEQ ID NO: 8, 9, 14, 17. 111 or 114
for TCR alpha
and SEQ ID NO: 10-13, 15-16, 18-19, 112-113 or 115-116 for TCR beta (as
further described
hereinbelow).
Sequence identity or homology can be determined using any protein or nucleic
acid
sequence alignment algorithm such as Blast, ClustalW, and MUSCLE.
The homolog may also refer to an ortholog, a deletion, insertion, or
substitution variant,
including an amino acid substitution, as further described hereinbelow.
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According to specific embodiments, the TCR alpha polypeptide and/or TCR beta
polypeptide may comprise conservative and/or non-conservative amino acid
substitutions. Non-
limiting examples of such substitution are known in the art and disclosed in
e.g. Haga-Friedman
A, et al. J Immunol. 2012; 188:5538-46; Froning K, et al. Nat Commun
[Internet]. Springer US;
5 2020; 11:1-14; Boulter JM, et al. Protein Eng. 2003; 16:707-11, the
contents of which arc fully
incorporated herein by reference.
According to specific embodiments, the TCR alpha poly-peptide and/or TCR beta
polypeptide do not comprise conservative and/or non-conservative amino acid
substitutions in an
endogenous cysteine residue e.g. in the endogenous cysteine residue located in
the hinge region of
10 each of the TCR alpha and beta.
The term "conservative substitution" as used herein, refers to the replacement
of an amino
acid present in the native sequence in the peptide with a naturally or non-
naturally occurring amino
or a peptidomimetics having similar steric properties. Where the side-chain of
the native amino
acid to be replaced is either polar or hydrophobic, the conservative
substitution should be with a
15 naturally occurring amino acid, a non-naturally occurring amino acid or
with a peptidomimetic
moiety which is also polar or hydrophobic (in addition to having the same
steric properties as the
side-chain of the replaced amino acid).
As naturally occurring amino acids are typically grouped according to their
properties,
conservative substitutions by naturally occurring amino acids can be easily
determined bearing in
20 mind the fact that in accordance with the invention replacement of
charged amino acids by
sterically similar non-charged amino acids are considered as conservative
substitutions.
For producing conservative substitutions by non-naturally occurring amino
acids it is also
possible to use amino acid analogs (synthetic amino acids) well known in the
art. A
peptidomimetic of the naturally occurring amino acid is well documented in the
literature known
to the skilled practitioner.
When affecting conservative substitutions the substituting amino acid should
have the
same or a similar functional group in the side chain as the original amino
acid.
The phrase "non-conservative substitutions" as used herein refers to
replacement of the
amino acid as present in the parent sequence by another naturally or non-
naturally occurring amino
acid, having different electrochemical and/or steric properties. Thus, the
side chain of the
substituting amino acid can be significantly larger (or smaller) than the side
chain of the native
amino acid being substituted and/or can have functional groups with
significantly different
electronic properties than the amino acid being substituted. Examples of non-
conservative
substitutions of this type include the substitution of phenylalanine or
cycohexylmethyl glycine for
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21
alanine, isoleucine for glycine, or -NH-CIV-CH2)5_C00KI-CO- for aspartic acid.
Those non-
conservative substitutions which fall under the scope of the present invention
are those which still
constitute a peptide having neuroprotective properties.
Thus, according to specific embodiments, one or more amino acid substitutions
(or changes
or mutations) in the TCR alpha are selected from the group consisting of
S116L, G119V and
F120L corresponding to the TCR alpha amino acid sequence as set forth in SEQ
ID NO: 9.
According to specific embodiments, the TCR alpha polypeptide comprises amino
acid
substitutions S1 16L, G1 19V and F120L corresponding to the human TCR alpha
polypeptide as
set forth in SEQ ID NO: 9
According to specific embodiments, the TCR alpha polypeptide comprises amino
acid
substitutions T48C, S116L, G119V and F120L corresponding to the human TCR
alpha
polypeptide as set forth in SEQ ID NO: 9, and the TCR beta polypeptide
comprises an amino acid
substitution 557C corresponding to the human TCR beta polypeptide as set forth
in SEQ ID NO:
12.
According to specific embodiments, the TCR alpha polypeptide and the TCR beta
polypeptide comprise a chimeric human and murine TCR alpha polypeptide and a
chimeric human
and murine TCR beta polypeptide; and further, the TCR alpha polypeptide
comprises amino acid
substitutions T48C corresponding to the human TCR alpha polypeptide as set
forth in SEQ ID
NO: 9, and the TCR beta polypeptide comprises an amino acid substitution S57C
corresponding
to the human TCR beta polypeptide as set forth in SEQ ID NO: 12.
According to specific embodiments, one or more amino acid changes in the TCR
alpha arc
selected from the group consisting of S21F, T32I, A72T corresponding to the
TCR alpha amino
acid sequence as set forth in SEQ ID NO: 9.
According to other specific embodiments, the TCR alpha polypeptide does not
comprise
one or more amino acid changes selected from the group consisting of S21F,
T32I, A72T
corresponding to the TCR alpha amino acid sequence as set forth in SEQ ID NO:
9.
According to specific embodiments, one or more amino acid changes in the TCR
beta are
selected from the group consisting of El8K, H23R, D39P, 554D corresponding to
the TCR beta
amino acid sequence as set forth in SEQ ID NO: 12.
According to other specific embodiments, the TCR beta polypeptide does not
comprise
one or more amino acid changes selected from the group consisting of E18K,
H23R, D39P, S54D
corresponding to the TCR beta amino acid sequence as set forth in SEQ ID NO:
12.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
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%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid sequence selected
form the group consisting of SEQ ID NO: 8, 14, 17, 20-23, 47, 111, 114 and 117-
120, each
possibility represents a separate embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence selected form the group consisting of SEQ ID NO: 8, 14, 17, 20-23,
47, 111, 114 and
117-120.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid sequence selected
form the group consisting of SEQ ID NO: 8, 14, 17 and 20-23, each possibility
represents a
separate embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence selected form the group consisting of SEQ ID NO: 8, 14, 17 and 20-23.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid sequence selected
form the group consisting of SEQ 1D NO: 14, 17, 20-23,47, 111, 114 and 117-
120, each possibility
represents a separate embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence selected form the group consisting of SEQ ID NO: 14, 17, 20-23, 47,
111, 114 and 117-
120.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid sequence selected
form the group consisting of SEQ ID NO: 14, 17 and 20-23, each possibility
represents a separate
embodiment of the present invention.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence selected form the group consisting of SEQ ID NO: 14, 17 and 20-23.
According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid sequence selected
form the group consisting of SEQ ID NO: 14 and 20-23, each possibility
represents a separate
embodiment of the present invention.
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According to specific embodiments, the TCR alpha polypeptide comprises an
amino acid
sequence selected form the group consisting of SEQ ID NO: 14 and 20-23.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid selected form the
group consisting of SEQ lD NO: 10-11, 15-16, 18-19,24-25, 112-113, 115-116 and
121-122, each
possibility represents a separate embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence selected form the group consisting of SEQ ID NO: 10-11, 15-16, 18-19,
24-25, 112-113,
115-116 and 121-122.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid selected form the
group consisting of SEQ 1D NO: 10-11, 15-16, 18-19 and 24-25, each possibility
represents a
separate embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence selected form the group consisting of SEQ 1D NO: 10-11, 15-16, 18-19
and 24-25.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid selected form the
group consisting of SEQ ID NO: 15-16, 18-19, 24-25, 112-113, 115-116 and 121-
122, each
possibility represents a separate embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence selected form the group consisting of SEQ ID NO: 15-16, 18-19, 24-25,
112-113, 115-
116 and 121-122.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence having at least 70 %, at least 80 % at least 85 %, at least 90 %, at
least 95 %, at least 96
%, at least 97 %, at least 98 %, at least 99 % or 100 % identity to an amino
acid selected form the
group consisting of SEQ ID NO: 15-16, 18-19 and 24-25, each possibility
represents a separate
embodiment of the present invention.
According to specific embodiments, the TCR beta polypeptide comprises an amino
acid
sequence selected form the group consisting of SEQ ID NO: 15-16, 18-19 and 24-
25.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 8
and the TCR beta polypeptide comprises SEQ ID NO: 10 or 11.
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According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 14
and the TCR beta polypeptide comprises SEQ ID NO: 15 or 16.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
111 and the TCR beta polypeptide comprises SEQ ID NO: 112 or 113.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 17
or 20 and the TCR beta polypeptide comprises SEQ ID NO: 18 or 19.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
114 or 117 and the TCR beta polypeptide comprises SEQ ID NO: 115 or 116.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 21
or 23 and the TCR beta polypeptide comprises SEQ ID NO: 24 or 25.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
118 or 120 and the TCR beta polypeptide comprises SEQ ID NO: 121 or 122.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 22
and the TCR beta polypeptide comprises SEQ ID NO: 15 or 16.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO:
119 and the TCR beta polypeptide comprises SEQ ID NO: 112 or 113.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 47
and the TCR beta polypeptide comprises SEQ ID NO: 18 or 19.
According to specific embodiments, the TCR alpha polypeptide comprises SEQ ID
NO: 47
and the TCR beta polypeptide comprises SEQ ID NO: 115 or 116.
According to specific embodiments, the TCR is devoid of an extracellular
domain
heterologous to the TCR alpha and beta polypeptides as defined herein.
However, according to specific embodiments, the TCR may comprise a
heterologous amino
acid sequence translationally fused to the TCR alpha and/or beta polypeptides,
as long as it is not
an extracellular domain (e.g. extracellular binding domain). Non-limiting
examples of such
heterologous sequences may comprise an amino acid sequence of a trafficking
sequence (e.g. a
CD8 membrane trafficking peptide such as provided in SEQ ID NO: 42), an
intracellular domain
such as a tag, a signaling domain and the like.
According to specific embodiments, the TCR may comprise a heterologous amino
acid
sequence translationally fused to the TCR alpha and/or beta polypeptides, as
long as it is not an
antigen-binding domain. Non-limiting examples of such heterologous sequences
may comprise an
amino acid sequence of a trafficking sequence (e.g. a CD8 membrane trafficking
peptide such as
provided in SEQ ID NO: 42), a receptor, a ligand or a hormone, a signaling
domain.
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According to specific embodiments, the TCR comprises a heterologous domain of
a
receptor or a ligand. Non-limiting examples of such receptors include EGFR,
HER2, VEGFR1,
VEGFR2, EpoR, FGFR1, FGFR2, FGFR3, FGFR4, MPL. CSF3R, CSF2RA, FLT3, CD117,
PD1,
CTLA4, CD28, Designed Ankyrin Repeat Protein (DARPin). Non-limiting examples
of such
5 ligands include TPO, 11 7, lL15, lL18, SCF.
According to specific embodiments, the TCR is devoid of a heterologous domain
of a
receptor or a ligand.
According to specific embodiments, the TCR comprises a heterologous signaling
domain,
which may be an activating or inhibitory signaling domain.
10
As used herein, the phrase "activating signal" refers to an amino acid
sequence capable of
transmitting a primary stimulatory signal or a co-stimulatory signal resulting
in T cell proliferation,
maturation, cytokine production and/or induction of regulatory or effector
functions. Typically, an
activating primary stimulatory signal domain comprises an ITAM domain and a co-
stimulatory
signal domain does not comprise an 1TAM domain.
15
Any known activating signal domain can be used with specific embodiments of
the present
invention. Non-limiting examples of activating signaling domains include the
intracellular
signaling domain of the proteins CD3zeta, FcR gamma, FcR beta, CD5, CD22,
CD79a, CD79b,
CD66d, 4-1BB, CD28, 0X40, 1COS, CD27, 1COS, G1TR, HVEM, T1M1, LFA1(CD11a),
CD2,
CD4OL, LIGHT, CD30, Fc receptor, DAP10 and DAP12.
20
As used herein the phrase "inhibitory signal" refers to an amino acid
sequence capable of
transmitting a primary or a secondary inhibitory signal resulting in
suppression of T cell
proliferation, maturation, cytokine production and/or induction of regulatory
or effector functions.
According to specific embodiments, the inhibitory signal domain is an
intracellular domain
comprising an domain.
25
Any known inhibitory signal domain can be used with specific embodiments of
the present
invention. Non-limiting examples of inhibitory signaling domains include the
intracellular
signaling domains of the proteins PD1, CTLA4, LAG3, TIM3, BTLA, TIGIT, 2B4,
CD300LF,
PECAM, LY9, SIRPA and CD244.
Methods of determining signaling of an activating or inhibitory signal in T
cells are well
known in the art and include, hut are not limited to, binding assay using e.g.
BiaCore, HPLC or
flow cytometry, enzymatic activity assays such as kinase activity assays, and
expression of
molecules involved in the signaling cascade using e.g. PCR, Western blot,
immunoprecipitation
and immunohistochemistry. Additionally, or alternatively, determining
transmission of a signal
can be effected by evaluating T cell activation or function. Methods of
evaluating T cell activation
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or function are well known in the art and include, but are not limited to,
proliferation assays such
as BRDU and thymidine incorporation, cytotwdcity assays such as chromium
release, cytokine
secretion assays such as intracellular cytokine staining ELISPOT and ELISA.
expression of
activation markers such as CD25, CD69 and CD69 using flow cytometry.
According to other specific embodiments, the TCR is devoid of a heterologous
signaling
domain.
According to specific embodiments, the TCR comprises a heterologous tag or is
bound to
a tag.
According to specific embodiments, the tag is a detectable moiety.
Examples of detectable moieties that can be used in the present invention
include but are
not limited to radioactive isotopes (such as [1251iodine). phosphorescent
chemiluminescent or
fluorescent chemicals and enzymes [e.g. horseradish perwddase (HPR), beta-
galactosidase and
alkaline phosphatase (AP)]. Further examples of detectable moieties include
those detectable by
Positron Emission Tomagraphy (PET) and Magnetic Resonance Imaging (MR1), all
of which are
well known to those of skill in the art.
Non-limiting examples of tags that can be used with specific embodiments
include chitin
binding protein (CBP)-tag, maltose binding protein (MBP)-tag, glutathione-S-
transferase (GST)-
tag, poly(His)-tag, FLAG tag, biotin, histidine, Epitope tags, such as, V5-
tag, c-myc-tag, and HA-
tag, and fluorescence tags such as green fluorescent protein (GFP or EGFP),
red fluorescent protein
(RFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), and
cyan fluorescent
protein (CFP); as well as derivatives of these tags, or any tag known in the
art.
Non-limiting schematic representations and sequences of the TCR of some
embodiments
of the invention are provided in Figures 2A-B and SEQ ID NOs: 2, 26-32, 46,
49, 63-68 and 125.
Typically, the TCR disclosed herein is produced by recombinant DNA technology.
Thus, according to an aspect of the present invention, there is provided at
least one
polynucleotide encoding the TCR or TCR complex.
As used herein the term "polynucleotide" refers to a single or double stranded
nucleic acid
sequence which is isolated and provided in the form of an RNA sequence, a
complementary
polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a
composite
polynucleotide sequences (e.g., a combination of the above).
According to specific embodiments, the at least one polynucleotide comprises a
nucleic
acid sequence encoding the TCR alpha polypeptide; and a nucleic acid sequence
encoding the TCR
beta polypeptide.
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According to specific embodiments, the TCR alpha and beta polypeptides are
encoded by
a single polynucleotide. Further description on expression of multiple
polypeptides from a single
polynucleotide is provided hereinbelow.
Thus, according to specific embodiments, the at least one polynucleotide is
one
polynucicotidc.
According to other specific embodiments, distinct polynucleotides arc used to
encode the
TCR alpha and beta polypeptides.
Thus, according to specific embodiments, the at least one polynucleotide is at
least two
polynucleotides.
According so specific embodiments, the at least one polynucleotide is two
polynucleotides.
According to specific embodiments, the at least one polynucleotide further
comprises a
nucleic acid sequence encoding a CD3 polypeptide.
To express any of the disclosed polypeptides in a cell, a polynucleotide
sequence encoding
the polypeptide is preferably ligated into a nucleic acid construct suitable
for cell expression. Such
a nucleic acid construct includes at least one cis-acting regulatory element
for directing expression
of the nucleic acid sequence. Cis-acting regulatory sequences include those
that direct constitutive
expression of a nucleotide sequence as well as those that direct inducible
expression of the
nucleotide sequence only under certain conditions. Thus, for example, a
promoter sequence for
directing transcription of the polynucleotide sequence in the cell in a
constitutive or inducible
manner is included in the nucleic acid construct.
The nucleic acid construct (also referred to herein as an "expression vector")
of some
embodiments of the invention includes additional sequences which render this
vector suitable for
replication and integration (e.g., shuttle vectors). In addition, a typical
cloning vectors may also
contain a transcription and translation initiation sequence, transcription and
translation terminator
and a polyadenylation signal. By way of example, such constructs will
typically include a 5' LTR,
a tRNA binding site, a packaging signal, an origin of second-strand DNA
synthesis, and a 3' LTR
or a portion thereof.
The nucleic acid construct of some embodiments of the invention typically
includes or
encodes a signal sequence for targeting the polypeptide to the cell surface.
According to a specific
embodiment, the signal sequence for this purpose is a mammalian signal
sequence or the signal
sequence of the polypeptide variants of some embodiments of the invention.
Eukaryotic promoters typically contain two types of recognition sequences, the
TATA box
and upstream promoter elements. The TATA box, located 25-30 base pairs
upstream of the
transcription initiation site, is thought to be involved in directing RNA
polymerase to begin RNA
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28
synthesis. The other upstream promoter elements determine the rate at which
transcription is
initiated.
Preferably, the promoter utilized by the nucleic acid construct of some
embodiments of the
invention is active in the specific cell population transformed, i.e. T cells.
Examples of T cell
specific promoters include lymphoid specific promoters [Calame et al.. (1988)
Adv. Immunol.
43:235-275]; in particular promoters of T-cell receptors [Winoto et al.,
(1989) EMBO J. 8:729-
733].
Enhancer elements can stimulate transcription up to 1,000 fold from linked
homologous or
heterologous promoters. Enhancers are active when placed downstream or
upstream from the
transcription initiation site. Many enhancer elements derived from viruses
have a broad host range
and are active in a variety of tissues. For example, the SV40 early gene
enhancer is suitable for
many cell types. Other enhancer/promoter combinations that are suitable for
some embodiments
of the invention include those derived from polyoma virus, human or murine
cytomegalovirus
(CMV), the long term repeat from various retroviruses such as murine leukemia
virus, murine or
Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold
Spring Harbor
Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by
reference.
In the construction of the expression vector, the promoter is preferably
positioned
approximately the same distance from the heterologous transcription start site
as it is from the
transcription start site in its natural setting. As is known in the art,
however, some variation in this
distance can be accommodated without loss of promoter function.
Polyadenylation sequences can also be added to the expression vector in order
to increase
the efficiency of mRNA translation. Two distinct sequence elements are
required for accurate and
efficient polyadenylation: GU or U rich sequences located downstream from the
polyadenylation
site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30
nucleotides
upstream. Termination and polyadenylation signals that are suitable for some
embodiments of the
invention include those derived from SV40.
In addition to the elements already described, the expression vector of some
embodiments
of the invention may typically contain other specialized elements intended to
increase the level of
expression of cloned nucleic acids or to facilitate the identification of
cells that carry the
recombinant DNA. For example, a number of animal viruses contain DNA sequences
that promote
the extra chromosomal replication of the viral genome in permissive cell
types. Plasrnids bearing
these viral replicons are replicated episomally as long as the appropriate
factors are provided by
genes either carried on the plasmid or with the genome of the host cell.
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The vector may or may not include a eukaryotic replicon. If a eukaryotic
replicon is
present, then the vector is amplifiable in eukaryotic cells using the
appropriate selectable marker.
If the vector does not comprise a eukaryotic replicon, no episomal
amplification is possible.
Instead, the recombinant DNA integrates into the genome of the engineered
cell, where the
promoter directs expression of the desired nucleic acid.
The expression vector of some embodiments of the invention can further include
additional
polynucleotide sequences that allow, for example, the translation of several
proteins from a single
mRNA such as an internal ribosome entry site (IRES) or a self-cleavable
peptide e.g. a 2A peptide
(e.g. P2A, T2A, E2A) which may be accompanied with a spacer and/or protease
e.g. furin cleavage
site (see e.g. Yang et al. Gene Ther. 2008; 15(21): 1411-1423); and sequences
for genomic
integration.
It will be appreciated that the individual elements comprised in the
expression vector can
be arranged in a variety of configurations. For example, enhancer elements,
promoters and the
like, and even the polynucleotide sequence(s) encoding the polypeptide can be
arranged in a "head-
to-tail" configuration, may be present as an inverted complement, or in a
complementary
configuration, as an anti-parallel strand. While such variety of configuration
is more likely to
occur with non-coding elements of the expression vector, alternative
configurations of the coding
sequence within the expression vector are also envisioned.
Examples for mammalian expression vectors include, but are not limited to,
pcDNA3,
pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto,
pCMV/myc/cyto,
pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from
Invitrogen, pCI which is available fromPromega, pMbac, pPbac, pBK-RSV and pBK-
CMV which
are available from Strategene, pTRES which is available from Clontech, and
their derivatives.
Expression vectors containing regulatory elements from eukaryotic viruses such
as
retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors
derived from
bovine papilloma virus include pBV-1MTHA. and vectors derived from Epstein Bar
virus include
pHEBO. and p205. Other exemplary vectors include pMSG. pAV009/A+. pMT010/A+.
pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of
proteins under the
direction of the SV-40 early promoter, SV-40 later promoter, metallothionein
promoter, murine
mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin
promoter, or other
promoters shown effective for expression in eukaryotic cells.
As described above, viruses are very specialized infectious agents that have
evolved, in
many cases, to elude host defense mechanisms. Typically, viruses infect and
propagate in specific
cell types. The targeting specificity of viral vectors utilizes its natural
specificity to specifically
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target predetermined cell types and thereby introduce a recombinant gene into
the infected cell.
The ability to select suitable vectors for transforming T cells is well within
the capabilities of the
ordinary skilled artisan and as such no general description of selection
consideration is provided
herein.
5
Recombinant viral vectors arc useful for in vivo expression of the
polypeptides since they
offer advantages such as lateral infection and targeting specificity. Lateral
infection is inherent in
the life cycle of, for example, retrovirus and is the process by which a
single infected cell produces
many progeny virions that bud off and infect neighboring cells. The result is
that a large area
becomes rapidly infected, most of which was not initially infected by the
original viral particles.
10
This is in contrast to vertical-type of infection in which the infectious
agent spreads only through
daughter progeny. Viral vectors can also be produced that are unable to spread
laterally. This
characteristic can be useful if the desired purpose is to introduce a
specified gene into only a
localized number of targeted cells.
Various methods can be used to introduce the expression vector of some
embodiments of
15
the invention into cells. Such methods are generally described in Sambrook
et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989.
1992), in
Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons,
Baltimore, Md.
(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
(1995), Vega et al.,
Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of
Molecular Cloning
20
Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.
[Biotechniques 4
(6): 504-512, 1986] and include, for example, stable or transient
transfection, lipofection,
electroporation and infection with recombinant viral vectors. In addition, see
U.S. Pat. Nos.
5,464,764 and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by viral infection offers several advantages
over other
25
methods such as lipofection and electroporation, since higher transfection
efficiency can be
obtained due to the infectious nature of viruses.
Currently preferred in vivo nucleic acid transfer techniques include
transfection with viral
or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I
virus, or adeno-associated
virus (AAV) and lipid-based systems. Useful lipids for lipid-mediated transfer
of the gene are. for
30
example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation,
14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are viruses,
most preferably
adenoviruses, AAV, lentiviruses, or retroviruses. A viral construct such as a
retroviral construct
includes at least one transcriptional promoter/enhancer or locus-defining
element(s), or other
elements that control gene expression by other means such as alternate
splicing, nuclear RNA
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export, or post-translational modification of messenger. Such vector
constructs also include a
packaging signal, long terminal repeats (LTRs) or portions thereof, and
positive and negative
strand primer binding sites appropriate to the virus used, unless it is
already present in the viral
construct. In addition, such a construct typically includes a signal sequence
for targeting the
polypeptide to the desired site in a cell. According to specific embodiments,
the signal sequence
comprises a membrane trafficking sequence. Such sequences are known in the
art. A non-limiting
example of such a sequence is the CD8 signal peptide such as provided in SEQ
ID No: 43.
Optionally, the construct may also include a signal that directs
polyadenylation, as well as one or
more restriction sites and a translation termination sequence. By way of
example, such constructs
will typically include a 5' LTR, a tRNA binding site, a packaging signal, an
origin of second-strand
DNA synthesis, and a 3' LTR or a portion thereof. Other vectors can be used
that are non-viral,
such as cationic lipids, polylysine, and dendrimers.
According to specific embodiments, the polynucleotide is expressed in the cell
while
knocking out e.g. an endogenous TCR (i.e. knock-in / knock-out). Such methods
are known in the
art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science
(1989) 244:1288-
1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814;
International Patent
Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent
Nos.
8771945, 8586526, 6774279 and UP Patent Application Publication Nos.
20030232410,
20050026157, US20060014264; the contents of which are incorporated by
reference in their
entireties] and include targeted homologous recombination, site specific
recombinases, PB
transposases and genome editing by engineered nucleases (e.g. meganucleases,
Zinc finger
nucleases (Z1-Ns), transcription-activator like effector nucleases (TALENs)
and CRISPR/Cas
system).
Non-limiting schematic representations and nucleic acid constructs encoding
the TCR of
some embodiments of the invention are provided in Figures 2A-B and SEQ ID NOs:
33-40, 53,
73-79 and 126.
Specific embodiments of the present invention also contemplate cells e.g. T
cells
comprising the TCR or TCR complex described herein and method of generating
same.
Thus, according to an aspect of the present invention, there is provided a
transduced cell
expressing the TCR complex or the TCR or at least one polynucleotide encoding
same.
According to an aspect of the present invention, there is provided a
transduced T cell
expressing the TCR complex or the TCR or at least one polynucleotide encoding
same.
According to an additional or an alternative aspect of the present invention,
there is
provided a method of producing a TCR or a TCR complex expressing cell, the
method comprising
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introducing into a cell the at least one polynucleotide encoding the TCR
disclosed herein, under
conditions which allow expression of the TCR or the TCR complex.
According to an additional or an alternative aspect of the present invention,
there is
provided a method of producing a TCR or a TCR complex expressing T cell, the
method
comprising introducing into a T cell the at least one polynucleotide encoding
the TCR disclosed
herein, under conditions which allow expression of the TCR or the TCR complex.
According to an additional or an alternative aspect of the present invention,
there is
provided a method of expressing a TCR or a TCR complex in a T cell, the method
comprising
introducing into a T cell the at least one polynucleotide encoding the TCR
disclosed herein, under
conditions which allow expression of the TCR or the TCR complex.
Such conditions may be for example an appropriate temperature (e.g., 37 C),
atmosphere
(e.g., air plus 5 % CO2), pH, light, medium, supplements and the like.
According to other specific embodiments, the introducing is effected in-vivo.
According to specific embodiments, the introducing is effected in-vitro or ex-
vivo.
Non-limiting Examples of cells into which the polynucleotide is introduced
include an
immune cell e.g. a T cell, a pluripotent stem cell, a hematopoietic stem and
progenitor cell and the
like.
Non-limiting examples of stem cells that can be used with specific embodiments
of the
invention include embryonic stern (ES) cells, gerrnline stem cells (GS cells),
embryonic germ cells
(EG cells), induced pluripotent stern (iPS) cells, cord blood-derived
pluripotent stem cells, bone
marrow-derived stem cells and the like.
According to a specific embodiment, the cell into which the polynucleotide is
introduced
is an iPS cell.
According to specific embodiments, the stem or progenitor cell may be further
differentiated to e.g. a T cell following the introducing to thereby obtain a
T cell expressing the
TCR disclosed herein. Methods of differentiating stem or progenitor cells into
T cells are known
in the art and disclosed e.g. in Blood, 105(4): 1431-1439, 2005; and
International Patent
Application Publication Nos. W02016/076415, W02017/221975).
According to specific embodiments, the cell into which the polynucleotide is
introduced is
a T cell.
As used herein, the term "T cell" refers to a differentiated lymphocyte
expressing CD3, and
includes CD4+ cells, CD8+ cells and NKT cells.
According to specific embodiments, the T cell is an effector cell.
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As used herein, the term "effector T cell" refers to a T cell that activates
or directs other
immune cells e.g. by producing cytokines or has a cytotoxic activity e.g.,
CD4+, Th1/Th2, CD8+
cytotwdc T lymphocyte.
According to specific embodiments, the T cell is a regulatory cell.
As used herein, the term -regulatory T cell" or -Treg" refers to a T cell that
negatively
regulates the activation of other T cells, including effector T cells, as well
as innate immune system
cells. Treg cells are characterized by sustained suppression of effector T
cell responses.
According to a specific embodiment, the Treg is a CD4+CD25+Foxp3+ T cell.
According to specific embodiments, the T cell is a CD4+ T cell.
According to other specific embodiments, the T cell is a CD8+ T cell.
According to specific embodiments, the T cell is a naïve T cell.
According to specific embodiments, the T cell is a memory T cell. Non-limiting
examples
of memory T cells include effector memory CD4+ T cells with a CD3+/CD4+/CD45RA-
/CCR7-
phenotype, central memory CD4+ T cells with a CD3+/CD4+/CD45RA-/CCR7+
phenotype,
effector memory CD8+ T cells with a CD3+/CD8+ CD45RA-/CCR7-phenotype and
central
memory CD8+ T cells with a CD3+/CD8+ CD45RA-/CCR7+ phenotype.
According to specific embodiments, the T cell is an NKT cell.
As used herein the term "NKT cell" refers to a specialized T cell that express
a variety of
molecular markers that are typically associated with NK cells, such as NK1.1.
NKT cells include
NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells.
According to other specific embodiments, the T cells is not an NKT cell.
Methods of obtaining T cells are well known in the art. Thus, for examples,
PBMCs can be
isolated by drawing whole blood from a subject and collection in a container
containing an anti-
coagulant (e.g. heparin or citrate); and apheresis. According to other
specific embodiments, the T
cells are obtained from a tissue comprising cells associated with a pathology.
Methods for
obtaining a tissue sample from a subject are well known in the art and include
e.g. biopsy, surgery
or necropsy and preparing a single cell suspension thereof. Following,
according to specific
embodiments, the T cell is enriched or purified from the peripheral blood or
from the single cell
suspension. There are several methods and reagents known to those skilled in
the art for purifying
T cells such as leukapheresis, sedimentation, density gradient centrifugation
(e.g. ficol I),
centrifugal elutriation, fractionation, chemical lysis of e.g. red blood cells
(e.g. by ACK), selection
of T cells using cell surface markers (using e.g. FACS sorter or magnetic cell
separation techniques
such as are commercially available e.g. from Invitrogen, Stemcell
Technologies, Cellpro,
Advanced Magnetics, or Miltenyi Biotec.), and depletion of other cell types by
methods such as
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eradication (e.g. killing) with specific antibodies or by affinity based
purification based on negative
selection (using e.g. magnetic cell separation techniques, FACS sorter and/or
capture ELISA
labeling). Such methods are described for example in THE HANDBOOK OF
EXPERIMENTAL
IMMUNOLOGY, Volumes 1 to 4, (D.N. Weir, editor) and FLOW CYTOMETRY AND CELL
SORTING (A. Radbruch, editor, Springer Verlag, 2000).
According to specific embodiments, the cell e.g. T cell is a mammalian cell.
According to specific embodiments, the cell e.g. T cell is a human cell.
According to specific embodiments, the cell e.g. T cell is of a healthy
subject.
According to specific embodiments, the cell e.g. T cell is of a subject
suffering from a
pathology (e.g. cancer).
According to specific embodiments, the cell e.g. T cell expresses an
endogenous CD3.
According to specific embodiments, the cell e.g. T cell expresses an exogenous
CD3.
The cell e.g. T cell of some embodiments of the invention is modified (or
engineered or
transduced) to upregulate or downregulate expression of a gene of interest
(e.g. endogenous TCR,
PD1, TGFBR1, B2M, CTLA4).
According to specific embodiments, the cell e.g. T cell does not express an
endogenous
TCR.
Thus, according to specific embodiments, the method further comprising
downregulating
expression of the endogenous TCR. Downregulation of the endogenous TCR may be
effected prior
to or subsequent to introducing to the cell the at least one polynucleotide
encoding the TCR
disclosed herein.
According to specific embodiments, the method further comprising
downregulating
expression of the endogenous TCR prior to introducing to the cell e.g. T cell
the at least one
polynucleotide encoding the TCR disclosed herein.
Methods of downregulating expression of a gene of interest e.g. an endogenous
TCR are
well known in the art [see for example W02019222275, W02015143224,
1JS20190388472; the
contents of which are incorporated by reference in their entireties] and
include targeted homologous
recombination, site specific recombinases, PB transposases and genome editing
by engineered
nucleases [Zinc finger nucleases (LI-Ns), transcription-activator like
effector nucleases (TALENs),
CRISPR/Cas system and constant shRNA]. Agents for introducing nucleic acid
alterations to a
TCR can be designed publically available sources or obtained commercially from
Transposagen,
Addgene and Sangamo Biosciences. A non-limiting example of a method of
downregulating an
endogenous TCR is described in details in the Examples section which follows.
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The cell e.g. T cell of some embodiments of the invention is modified (or
engineered or
transduced) to express a chimeric receptor, e.g. a chimeric antigen receptor
(CAR).
As used herein, the phrase "transduced with a CAR" or "genetically engineered
to express
a CAR" refers to cloning of a nucleic acid sequence encoding a chimeric
antigen receptor (CAR),
5
wherein the CAR comprises an antigen recognition moiety of an antibody and a
T-cell activation
moiety. A chimeric antigen receptor (CAR) is an artificially constructed
hybrid protein or
polypeptide containing an antigen binding domain of an antibody (e.g., a
single chain variable
fragment (scFv)) linked to T-cell signaling or T-cell activation domains.
Method of transducing
with a CAR are known in the art and are disclosed e.g. in Davila et al.
Oncoimmunology. 2012
10
Dec 1;1(9):1577-1583; Wang and Riviere Cancer Gene Ther. 2015 Mar;22(2):85-
94); Maus et al.
Blood. 2014 Apr 24;123(17):2625-35; Porter DL The New England journal of
medicine. 2011,
365(8):725-733; Jackson HJ, Nat Rev Clin Oncol. 2016;13(6):370-383; and
Globerson-Levin et
al. Mol Ther. 2014;22(5):1029-1038. According to specific embodiments, the
antigen recognition
moiety is specific for a pathologic cell.
15
According to other specific embodiments, the cell is not transduced (i.e.
does not express)
a CAR.
According to specific embodiments, the cells e.g. T cells can be freshly
isolated, stored
e.g., cryopreserved (i.e. frozen) at e.g. liquid nitrogen temperature at any
stage for long periods of
time (e.g., months, years) for future use; and cell lines.
20
Methods of cryopreservation are commonly known by one of ordinary skill in
the art and
are disclosed e.g. in International Patent Application Publication Nos.
W02007054160 and WO
2001039594 and US Patent Application Publication No. U520120149108.
According to specific embodiments, the cells e.g. T cells can be stored in a
cell bank or a
depository or storage facility.
25
Consequently, the present teachings further suggest the use of the cells
e.g. T cells and the
methods disclosed herein as, but not limited to, a source for adoptive cells
therapies.
Thus, according to an aspect of the present invention, the cells e.g. T cells
disclosed herein
are for use in adoptive cell therapy.
The cells e.g. T cells used according to specific embodiments of the present
invention may
30
he autologous or non-autologous; they can he syngeneic or non-syngeneic:
allogeneic or
xenogeneic to the subject; each possibility represents a separate embodiment
of the present
invention.
According to specific embodiments, the cells are autologous to the subject.
According to specific embodiments, the cells are non-autologous to the
subject.
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36
According to specific embodiments, the cells are allogeneic to the subject.
As the T cells of some embodiments do not express an endogenous TCR and
express a
TCR or TCR complex devoid of a binding domain (an antigen-binding domain, a
heterologous
extracellular binding domain), the T cells of some embodiments do not
stimulate a graft versus
host disease in a non-autologous subject.
According to specific embodiments, the T cells described herein arc cultured,
expanded
and/or activated ex-vivo prior to administration to the subject.
Methods of culturing, expanding and activating T cells are well known to the
skilled in the
art. For example, the T cells of some embodiments may be expanded ex-vivo in
the presence of
an anti-CD3 antibody, an anti-CD28 antibody, anti-CD3 and anti-CD28 coated
beads (such as the
CD3CD28 MACSiBeads obtained from Miltenyi Biotec) with or without IL-2.
Since the TCR or TCR complex disclosed herein are devoid of a binding domain
(an
antigen-binding domain, a heterologous extracellular binding domain), the T
cells of some
embodiments of the invention may be administered to a subject in combination
with a therapeutic
composition directed for binding a target cell (e.g. a pathological cell) on
the one hand and the TCR
complex on the other hand, thereby activating the T cell towards the target
cell.
Thus, according to an aspect of the present invention, there is provided a
method of treating
a disease associated with a pathological cell in a subject in need thereof,
the method comprising
administering to the subject a therapeutically effective amount of the T cell
disclosed herein; and
a therapeutic composition capable of binding the pathological cell and the TCR
complex, thereby
treating the disease in the subject.
According to an additional or an alternative aspect of the present invention,
there is
provided the T cell disclosed herein; and a therapeutic composition capable of
binding a
pathological cell and the TCR complex, for use in treating a disease
associated with the
pathological cell in a subject in need thereof.
As used herein, the term "subject" includes mammals, preferably human beings
at any age
and of any gender. According to specific embodiments, the term -subject"
refers to a subject who
suffers from the pathology (disease, disorder or medical condition). According
to specific
embodiments, this term encompasses individuals who are at risk to develop the
pathology.
As used herein the term "treating" refers to curing, reversing, attenuating,
alleviating,
minimizing, suppressing or halting the deleterious effects of a disease or
disorder (e.g. cancer).
Those of skill in the art will understand that various methodologies and
assays can be used to assess
the development of a pathology, and similarly, various methodologies and
assays may be used to
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37
assess the reduction, remission or regression of a pathology (e.g. a
malignancy), as discussed
below.
As used herein, the term "preventing" refers to keeping a disease, disorder or
condition
from occurring in a subject who may be at risk for the disease, but has not
yet been diagnosed as
having the disease.
As used herein the phrase, -disease associated with a pathological cell" means
that
pathologic cells drive onset and/or progression of the disease.
According to specific embodiments, the disease can benefit from modulating
immune cells.
As used herein the phrase "a disease that can benefit from modulating immune
cells" refers
to diseases in which the subject's immune response activity may be sufficient
to at least ameliorate
symptoms of the disease or delay onset of symptoms, however for any reason the
activity of the
subject's immune response in doing so is less than optimal.
According to specific embodiments, the disease can benefit from activating
immune cells.
Non-limiting examples of diseases that can benefit from activating immune
cells include
hyper-proliferative diseases, diseases associated with immune suppression,
immunosuppression
caused by medication (e.g. mTOR inhibitors, calcineurin inhibitor, steroids)
and infections.
According to specific embodiments, the disease comprises an infection.
As used herein, the term "infection" or "infectious disease" refers to a
disease induced by
a pathogen. Specific examples of pathogens include, viral pathogens, bacterial
pathogens e.g.,
intracellular mycobacterial pathogens (such as, for example, Mycobacterium
tuberculosis),
intracellular bacterial pathogens (such as, for example, Listeria
monocytogenes), or intracellular
protozoan pathogens (such as, for example, Leishmania and Trypanosoma).
Specific types of viral pathogens causing infectious diseases include, but are
not limited
to, retroviruses, circoviruses, parvoviruses, papovaviruses, adenoviruses,
herpesviruses,
iridoviruses, poxviruses, hepadnaviruses, picornaviruses, caliciviruses,
togaviruses, flaviviruses,
reoviruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses,
coronaviruses,
arenaviruses, and filoviruses.
Specific examples of viral infections which may be treated according to
specific
embodiments of the present invention include, but are not limited to, human
immunodeficienc y
virus (HIV)-induced acquired immunodeficiency syndrome (AIDS), influenza,
rhinoviral
infection, viral meningitis, Epstein-Barr virus (EBV) infection, hepatitis A,
B or C virus infection,
measles, papilloma virus infection/warts, cytomegalovirus (CMV) infection,
Herpes simplex virus
infection, yellow fever, Ebola virus infection, rabies, etc.
According to specific embodiments, the disease comprises a hyper-proliferative
disease.
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According to specific embodiments, the hyper-proliferative disease comprises
sclerosis,
fibrosis, Idiopathic pulmonary fibrosis, psoriasis, systemic
sclerosis/scleroderma, primary biliary
cholangitis, primary sclerosing cholangitis, liver fibrosis, prevention of
radiation-induced
pulmonary fibrosis, myelofibrosis or retroperitoneal fibrosis.
According to other specific embodiments, the hyper-proliferative disease
comprises
cancer.
Thus, according to specific embodiments the pathological cell is a cancerous
cell.
Cancers which may be treated by some embodiments of the invention can be any
solid or
non-solid tumor, cancer metastasis and/or a pre-cancer.
According to specific embodiments, the cancer is a malignant cancer.
Examples of cancer include but are not limited to, carcinoma, blastoma,
sarcoma and
lymphoma. More particular examples of such cancers include, but are not
limited to, tumors of the
gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal
carcinoma, colorectal cancer,
colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis
type 2, hereditary
nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer,
hereditary nonpolyposis
type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with
esophageal cancer,
stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors),
endometrial carcinoma,
dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors,
prostate cancer,
prostate adenocarcinorna, renal cancer (e.g., Wilms' tumor type 2 or type 1),
liver cancer (e.g.,
hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer), bladder
cancer, embryonal
rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cells
tumor, immature
teratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor,
choriocarcinoma, placental
site trophoblastic tumor, epithelial adult tumor, ovarian carcinoma, serous
ovarian cancer, ovarian
sex cord tumors, cervical carcinoma, uterine cervix carcinoma, small-cell and
non-small cell lung
carcinoma, nasopharyngeal, breast carcinoma (e.g., ductal breast cancer,
invasive intraductal
breast cancer, sporadic; breast cancer, susceptibility to breast cancer, type
4 breast cancer, breast
cancer-1, breast cancer-3; breast-ovarian cancer), squamous cell carcinoma
(e.g., in head and neck),
neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphomas
(e.g., Hodgkin's
disease, non-Hodgkin's lymphoma, B cell, Burkitt, cutaneous T cell,
histiocytic, lymphoblastic, T
cell, thymic), gliomas, adenocarcinoma, adrenal tumor, hereditary
adrenocortical carcinoma, brain
malignancy (tumor), various other carcinomas (e.g., bronchogenic large cell,
ductal, Ehrlich-Lettre
ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat
cell, small cell, spindle
cell, spinocellular, transitional cell, undifferentiated, carcinosarcoma,
choriocarcinoma,
cystadenocarcinoma), ependimoblastoma, epithelioma, erythroleukemia (e.g.,
Friend,
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lymphoblast), fibrosarcoma, giant cell tumor, glial tumor. glioblastoma (e.g.,
multiforme,
astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma,
hybridoma (e.g.,
B cell), hypemephroma, insulinoma, islet tumor, keratoma, leiomyoblastoma,
leiomyosarcoma,
leukemia (e.g., acute lymphatic, acute lymphoblastic, acute lymphoblastic pre-
B cell, acute
lymphoblastic T cell leukemia, acute - mcgakaryoblastic, monocytic, acute
myclogenous, acute
myeloid, acute myeloid with cosinophilia, B cell, basophilic, chronic myeloid,
chronic, B cell,
eo si nophi 1 i c , Friend, granulocytic or myelocytic, hairy cell,
lymphocytic, megakaryoblasti c ,
monocytic, monocytic-macrophage, myeloblastic, myeloid, myelomonocytic, plasma
cell, pre-B
cell, promyelocytic, subacute, T cell, lymphoid neoplasm, predisposition to
myeloid malignancy,
acute nonlymphocytic leukemia), lympho sarcoma, melanoma, mammary tumor,
mastocyto ma,
medulloblastoma, me sothelioma, metastatic tumor, monocyte tumor, multiple
myelo ma ,
myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue glial tumor,
nervous tissue
neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma, osteochondroma,
osteomyelo ma,
osteosarcoma (e.g., Ewing's). papilloma, transitional cell, pheochromocytoma,
pituitary tumor
(invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g.,
Ewing's, histiocytic
cell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneous tumor,
teratocarcinoma (e.g.,
pluripotent), teratoma, testicular tumor, thymoma and trichoepithelioma,
gastric cancer,
fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni
syndrome,
liposarcorna, lynch cancer family syndrome 11, male germ cell tumor, mast cell
leukemia,
medullary thyroid, multiple meningioma, endocrine neoplasia myxosarcoma,
paraganglioma,
familial nonchromaffin, pilomatricoma, papillary, familial and sporadic,
rhabdoid predisposition
syndrome, familial, rhabdoid tumors, soft tissue sarcoma, and Turcot syndrome
with glioblastoma.
According to specific embodiments, the cancer is a pre-malignant cancer.
Pre-cancers are well characterized and known in the art (refer, for example,
to Berman JJ.
and Henson DE., 2003. Classifying the pre-cancers: a metadata approach. BMC
Med Inform Decis
Mak. 3:8). Examples of pre-cancers include, but are not limited to, acquired
small pre-cancers,
acquired large lesions with nuclear atypia, precursor lesions occurring with
inherited hyperplastic
syndromes that progress to cancer, and acquired diffuse hyperplasias and
diffuse metaplasias.
Non-limiting examples of small pre-cancers include HGSIL (High grade squamous
intraepithelial
lesion of uterine cervix), AIN (anal intraepithelial neoplasia), dysplasia of
vocal cord, aberrant
crypts (of colon), PIN (prostatic intraepithelial neoplasia).
Non-limiting examples of acquired large lesions with nuclear atypia include
tubular
adenoma, AlLD (angioimmunoblastic lymphadenopathy with dysproteinemia),
atypical
meningioma, gastric polyp, large plaque parapsoriasis, myelodysplasia,
papillary transitional cell
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carcinoma in-situ, refractory anemia with excess blasts, and Schneiderian
papilloma. Non-limiting
examples of precursor lesions occurring with inherited hyperplastic syndromes
that progress to
cancer include atypical mole syndrome, C cell adenomatosis and MEA. Non-
limiting examples
of acquired diffuse hyperplasias and diffuse metaplasias include Paget's
disease of bone and
5 ulcerative colitis.
According to specific embodiments, the cancer is selected from the group
consisting of
lymphoma, leukemia, glioblastoma, colon cancer, gastric cancer, pancreatic
cancer, ovarian
cancer, lung cancer and skin cancer.
According to specific embodiments, the disease can benefit from inhibiting
immune cells.
10 According to specific embodiments, the disease is an autoimtnune
disease. Such
autoimmune diseases include, but are not limited to, cardiovascular diseases,
rheumatoid diseases,
glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic
diseases, neurological
diseases, muscular diseases, nephric diseases, diseases related to
reproduction, connective tissue
diseases and systemic diseases.
15
Examples of autoimmune cardiovascular diseases include, but are not
limited to
atherosclerosis (Matsuura E. et at., Lupus. 1998;7 Suppl 2:S135), myocardial
infarction (Vaarala 0.
Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et at., Lupus 1998;7 Suppl
2:S107-9),
Wegener's uanulomatosis, Takayasu's arteritis, Kawasaki syndrome (Praprotnik
S. et at., Wien
Klin Wochenschr 2000 Aug 25;112 (15-16):660), anti-factor VM autoimmune
disease (Lacroix-
20
Desmazes S. et al., Setnin Thromb Hemost.2000;26 (2):157), necrotizing
small vessel vasculitis,
microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal
necrotizing and
crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May;151
(3):178),
antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999;14
(4):171), antibody-induced
heart failure (Wallukat G. et at., Am J Cardiol. 1999 Jun 17;83 (12A):75H),
thrombocytopenic
25
purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun;14 (2):114; Semple JW.
et at., Blood 1996 May
15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk
Lymphoma 1998 Jan;28
(3-4):285; Sallah S. et al., Ann Hematol 1997 Mar;74 (3):139), cardiac
autoimmunity in Chagas'
disease (Cunha-Neto E. et at., J Clin Invest 1996 Oct 15;98 (8):1709) and anti-
helper T lymphocyte
autoimmunity (Caporossi AP. et at., Viral Immunol 1998;11 (1):9).
30
Examples of autoimmune rheumatoid diseases include, hut are not limited to
rheumatoid
arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791; Tisch R,
McDevitt HO. Proc Natl
Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan
Vosvvinkel et at.,
Arthritis Res 2001; 3 (3): 189).
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Examples of autoimmune glandular diseases include, but are not limited to,
pancreatic
disease, Type I diabetes, thyroid disease, Graves' disease, thyroiditis,
spontaneous autoimmune
thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian
autoimmunity, autoimmune anti-
sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular
syndrome. diseases
include, but arc not limited to autoimmunc diseases of the pancreas, Type 1
diabetes (Castano L. and
Eiscnbarth GS. Aim. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract
1996 Oct;34
Suppl:S125), autoimmune thyroid diseases, Graves' disease (Orgiazzi J.
Endocrinol Metab Clin
North Am 2000 Jun;29 (2):339; Sakata S. et at., Mol Cell Endocrinol 1993
Mar;92 (1):77),
spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000
Dec 15;165
(12):7262), Hashimoto's thyroiditis (Toyoda N. et at., Nippon Rinsho 1999
Aug;57 (8):1810),
idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8):1759), ovarian
autoimmunity
(Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm
infertility
(Diekman AB. et at., Am J Reprod Immunol. 2000 Mar;43 (3):134), autoimmune
prostatitis
(Alexander RB. et at., Urology 1997 Dec;50 (6):893) and Type 1 autoimmune
polyglandular
syndrome (Hara T. et al., Blood. 1991 Mar 1;77 (5):1127).
Examples of autoimmune gastrointestinal diseases include, but are not limited
to, chronic
inflammatory intestinal diseases (Garcia Herola A. et at., Gastroenterol
Hepatol. 2000 Jan;23
(1):16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138
(2):122), colitis,
ileitis and Crohn's disease.
Examples of autoimmune cutaneous diseases include, but are not limited to,
autoimmune
bullous skin diseases, such as, but are not limited to, pemphigus vulgaris,
bullous pemphigoid and
pemphigus foliaceus.
Examples of autoimmune hepatic diseases include, but are not limited to,
hepatitis,
autoimmune chronic active hepatitis (Franco A. et at., Clin Immunol
Immunopathol 1990 Mar;54
(3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91
(5):551; Strassburg CP.
et at., Eur J Gastroenterol Hepatol. 1999 Jun;11 (6):595) and autoimmune
hepatitis (Maims MP. J
Hepatol 2000 Aug;33 (2):326).
Examples of autoimmune neurological diseases include, but are not limited to,
multiple
sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1;112 (1-2):1),
Alzheimer's disease (Oron L.
etal., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And
Kraig E, Int Rev
Immunol 1999;18 (1-2):83; Oshima M. etal., Eur JImmunol 1990 Dec;20
(12):2563), neuropathies,
motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3):191);
Guillain-Barre syndrome
and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234),
myasthenia,
Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319
(4):204);
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42
paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic
cerebellar atrophy and stiff-
man syndrome (Hiemstra HS. et at., Proc Natl Acad Sci units S A 2001 Mar 27;98
(7):3988); non-
paraneoplastic stiff man syndrome, progressive cerebellar atrophies,
encephalitis, Rasmussen' s
encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la
Tourette syndrome and
autoimmunc polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol
(Paris) 2000 Jan;156
(1):23); dysimmunc neuropathics (Nobilc-Orazio E. et at., Electrocncephalogr
Clin Neurophysiol
Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita
(Vincent A. et at.,
Ann NY Acad Sci. 1998 May 13;841:482), neuritis, optic neuritis (Soderstrom M.
et al., J Neurol
Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.
Examples of autoimmune muscular diseases include, but are not limited to,
myositis,
autoimmune myositis and primary Sjogren's syndrome (Feist E. et at., Int Arch
Allergy Immunol
2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. etal.,
Biomed Pharmacother
1999 Jun;53 (5-6):234).
Examples of autoimmune nephric diseases include, but are not limited to,
nephritis and
autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;1
(2):140).
Examples of autoimmune diseases related to reproduction include, but are not
limited to,
repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S107-9).
Examples of autoimmune connective tissue diseases include, but are not limited
to, ear
diseases, autoimmune ear diseases (Yoo TJ. et at., Cell Immunol 1994 Aug;157
(1):249) and
autoimmune diseases of the inner ear (Gloddek B. et at., Ann N Y Acad Sci 1997
Dec 29;830:266).
Examples of autoimmune systemic diseases include, but are not limited to,
systemic lupus
erythematosus (Erikson J. et al., Immunol Res 1998;17 (1-2):49) and systemic
sclerosis
(Renaudineau Y. et at., Clin Diagn Lab Immunol. 1999 Mar;6 (2):156); Chan OT.
et at., Immunol
Rev 1999 Jun;169:107).
According to specific embodiments, the disease is graft rejection disease.
Examples of diseases associated with transplantation of a graft include, but
are not limited
to, graft rejection, chronic graft rejection, subacute graft rejection,
hyperacute graft rejection, acute
graft rejection and graft versus host disease.
According to specific embodiments, the disease is an allergic disease.
Examples of allergic diseases include, hut are not limited to, asthma, hives,
urticaria, pollen
allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy,
chemical allergy, drug
allergy, insect bite allergy, animal dander allergy, stinging plant allergy,
poison ivy allergy and food
allergy.
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As mentioned, according to specific embodiments, the T cells are administered
to the
subject in combination with a therapeutic composition specific for the
pathological cell [e.g. binds
an antigen overexpressed or solely expressed by a pathologic (e.g. cancerous)
cell as compared to
a non-pathologic cell] on the one hand and capable of binding the TCR complex
on the other hand.
According to specific embodiments, the therapeutic composition binds CD3.
According to specific embodiments, the therapeutic composition binds the
constant or the
hinge region of the TCR.
According to specific embodiments, the therapeutic composition hinds a
heterologous tag
fused to the TCR complex or a tag bound to the TCR complex. Non-limiting
Examples of such
tags are known in the art and are further described in details hereinabove.
The administration of the T cells and the administration of the therapeutic
composition can
be effected in the same route or in separate routes.
The administration of the T cells may be prior to, following or concomitant
with the
therapeutic.
Multiple rounds of administration of the T cells and/or the therapeutic
composition can be
administered.
According to specific embodiments, multiple distinct therapeutic compositions
specific for
the pathological cell (e.g. targeting different antigens) and capable of
binding the TCR complex
are provided to the subject.
Therapeutic compositions capable of binding a pathological cell and the TCR
complex are
known in the art and include, but not limited to, antibodies such as hi-
specific and tri-specific
antibodies.
The term "antibody" as used herein includes intact molecules as well as
functional
fragments thereof (that are capable of binding to an epitope of an antigen).
As used herein, the term "epitope" refers to any antigenic determinant on an
antigen to
which the paratope of an antibody binds. Epitopic determinants usually consist
of chemically active
surface groupings of molecules such as amino acids or carbohydrate side chains
and usually have
specific three dimensional structural characteristics, as well as specific
charge characteristics.
According to a specific embodiment, the antibody fragments include, but are
not limited to,
single chain, Fah, Fah' and F(ah`),) fragments, Fd, Fcah, Fv, dsFv, scFvs,
diahodies, mini bodies,
nanobodies, Fab expression library or single domain molecules such as VH and
VL that are capable
of binding to an epitope of the antigen in an HLA restricted manner.
According to specific embodiments, the therapeutic compositions is at least a
hi-specific
antibody.
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As used herein, the term "hi-specific antibody" refers to an antibody having
two distinct
antigen-binding moieties, wherein the first binding moiety has affinity to the
TCR complex and
the second binding moiety has affinity for an antigen distinct from the TCR
complex. According
to specific embodiments, the hi-specific antibody binds the TCR complex on the
one hand and an
antigen expressed by a pathological cell (e.g. cancerous cell) on the other
hand.
Methods of producing bi-specific antibodies arc known in the art and disclosed
for
examples in US Patent Numbers 4,474,893, 5,959,084, US and 7,235,641,
7,183,076, U.S.
Publication Number 20080219980 and International Publication Numbers WO
2010/115589,
W02013150043 and W02012118903 all incorporated herein by their entirety; and
include, for
example, chemical cross-linking (Brennan, et al., Science 229,81 (1985); Raso,
et al., J. Biol.
Chern. 272, 27623 (1997)), disulfide exchange, production of hybrid-hybridomas
(quadromas), by
transcription and translation to produce a single polypeptide chain embodying
a hi-specific
antibody, or by transcription and translation to produce more than one
polypeptide chain that can
associate covalently to produce a hi-specific antibody. The contemplated hi-
specific antibody can
also be made entirely by chemical synthesis.
Hence, according to specific embodiments, the therapeutic antibody comprises
at least one
arm that binds an antigen overexpressed or solely expressed by a pathological
cell and one arm
comprising an antibody moiety that binds the TCR complex.
Selection of the therapeutic antibody used is well within the capability of
those skilled in
the art, and depends on the type of the disease and the antigens expressed by
the pathologic cells
associated with the pathology.
According to specific embodiments, the therapeutic composition comprises an
anti-CD3
antibody.
According to specific embodiments, the anti-CD3 antibody moiety is of an
antibody
selected from the group consisting of L2K, TR66, OKT3 UCHT1, humanized UHCT1,
F6A, SP34
and I2C.
According to specific embodiments, the anti-CD3 antibody moiety is of an
antibody
selected from the group consisting of L2K, TR66 and OKT3.
According to specific embodiments, the anti-CD3 antibody moiety is of an
antibody
selected from the group consisting of L2K, SP34 and UCHT1.
According to specific embodiments, the therapeutic antibody binds an antigen
expressed
by cancerous cells. Non-limiting examples of such antigens include CD19, EGFR,
HER2, MUC-
1, CA-125, mesothelin, ROR1. GPC3, PSCA, CD133, CD70, EpCAM, CEA, CAIX, CD171,
GD2, Tn-MUCL EGFRvBI, ADGRE2, CD33, CD123, CCR1, CLEC12A, LILRB2, BCMA,
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CD138, gp100 and the antigens disclosed in Q He et al. (2019) J Hematol Oncol
12, 99, the
contents of which are fully incorporated herein by reference.
According to some embodiments of the invention, the therapeutic antibody is
selected from
the group consisting of Blinatumomab, AMG 330, AMG701, Orlotamab, AMG420, CC-
93269
5 APV0436, JNJ-63709178, AMG757, MT110, Tebentafusp, Odronextamab, RG6007,
RG6194,
RG6232, RG7828, Tcclistamab, Mosunctuzumab, RG7802 cibisatamab, ccvostamab,
Glotitamab
and 1MMTAC (e.g. RG6290).
According to specific embodiments, the pathological cells expresses CD19 and
the
therapeutic composition comprises Blinatumomab.
10
According to specific embodiments, the pathological cell expresses
EpCAM and said
therapeutic composition comprises MT110.
According to specific embodiments, the pathological cell expresses CD20 and
said
therapeutic composition comprises Odronextamab.
According to specific embodiments, the pathological cell expresses CD20 and
said
15 therapeutic composition comprises Mosunetuzumab.
According to specific embodiments, the pathological cell expresses HLA-A2-WT1
and
said therapeutic composition comprises RG6007.
According to specific embodiments, the pathological cell expresses HER2 and
said
therapeutic composition comprises RG6194.
20 According to specific embodiments, the pathological cell expresses
TYRP1 and said
therapeutic composition comprises RG6232.
According to specific embodiments, the pathological cell expresses MAGE-A4 and
said
therapeutic composition comprises RG6290.
According to specific embodiments, the therapeutic composition comprises an
anti-TCR
25 antibody.
According to specific embodiments, the anti-TCR antibody moiety is of an
antibody clone
selected from the group consisting of 1P26,
WT31. T10B9,
BW242/41, 8A3, 3A8, Jovi-1.
According to specific embodiments, the T cells and the therapeutic
compositions capable
30 of binding the pathological cell and the TCR complex can he
administered to a subject in
combination with other established or experimental therapeutic regimen to
treat a disease
associated with pathologic cells (e.g. cancer) including, but not limited to
analgesics,
chemotherapeutic agents, radiotherapeutic agents, cytotoxic therapies
(conditioning), hormonal
therapy and other treatment regimens (e.g., surgery) which are well known in
the art.
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The T cells disclosed herein and/or the therapeutic compositions capable of
binding the
pathological cell and the TCR complex disclosed herein can be administered to
the subject per se,
or in a pharmaceutical composition where it is mixed with suitable carriers or
excipients.
As used herein a "pharmaceutical composition" refers to a preparation of one
or more of
the active ingredients described herein with other chemical components such as
physiologically
suitable carriers and excipients. The purpose of a pharmaceutical composition
is to facilitate
administration of a compound to an organism.
Herein the term "active ingredient" refers to the T cells and/or the
therapeutic compositions
capable of binding the pathological cell and the TCR complex accountable for
the biological effect.
Thus, according to specific embodiments, the T cells are the active ingredient
in the
formulation.
Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically
acceptable carrier" which may be interchangeably used refer to a carrier or a
diluent that does not
cause significant irritation to an organism and does not abrogate the
biological activity and
properties of the administered compound. An adjuvant is included under these
phrases.
Herein the term "excipient" refers to an inert substance added to a
pharmaceutical
composition to further facilitate administration of an active ingredient.
Examples, without
limitation, of excipients include calcium carbonate, calcium phosphate,
various sugars and types
of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene
glycols.
Techniques for formulation and administration of drugs may be found in
"Remington' s
Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition,
which is incorporated
herein by reference.
Suitable routes of administration may, for example, include oral, rectal,
transmucosal,
especially transnasal, intestinal or parenteral delivery, including
intramuscular, intradermal,
subcutaneous and intramedullary injections as well as intrathecal, direct
intraventricular,
intracardiac, e.g., into the right or left ventricular cavity, into the common
coronary artery,
intravenous, intraperitoneal, intranasal, or intraocular injections.
Conventional approaches for drug delivery to the central nervous system (CNS)
include:
neurosurgical strategies (e.g., intracerebral injection or
intracerebroventricular infusion); molecular
manipulation of the agent (e.g., production of a chimeric fusion protein that
comprises a transport
peptide that has an affinity for an endothelial cell surface molecule in
combination with an agent
that is itself incapable of crossing the BBB) in an attempt to exploit one of
the endogenous transport
pathways of the BBB; pharmacological strategies designed to increase the lipid
solubility of an
agent (e.g., conjugation of water-soluble agents to lipid or cholesterol
carriers); and the transitory
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47
disruption of the integrity of the BBB by hyperosmotic disruption (resulting
from the infusion of
a mannitol solution into the carotid artery or the use of a biologically
active agent such as an
angiotensin peptide). However, each of these strategies has limitations, such
as the inherent risks
associated with an invasive surgical procedure, a size limitation imposed by a
limitation inherent
in the endogenous transport systems, potentially undesirable biological side
effects associated with
the systemic administration of a chimeric molecule comprised of a carrier
motif that could be
active outside of the CNS, and the possible risk of brain damage within
regions of the brain where
the BBB is disrupted, which renders it a suboptimal delivery method.
Alternately, one may administer the pharmaceutical composition in a local
rather than
systemic manner, for example, via injection of the pharmaceutical composition
directly into a tissue
region of a patient.
According to a specific embodiment, the T cells of the invention or the
pharmaceutical
composition comprising same is administered via an IV route.
Pharmaceutical compositions of some embodiments of the invention may be
manufactured
by processes well known in the art, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.
Pharmaceutical compositions for use in accordance with some embodiments of the
invention thus may be formulated in conventional manner using one or more
physiologically
acceptable carriers comprising excipients and auxiliaries, which facilitate
processing of the active
ingredients into preparations which, can be used pharmaceutically. Proper
formulation is
dependent upon the route of administration chosen.
For injection, the active ingredients of the pharmaceutical composition may be
formulated
in aqueous solutions, preferably in physiologically compatible buffers such as
Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants
appropriate to the barrier to be permeated are used in the formulation. Such
penetrants are generally
known in the art.
For oral administration, the pharmaceutical composition can be formulated
readily by
combining the active compounds with pharmaceutically acceptable carriers well
known in the art.
Such carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral
ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid excipient,
optionally grinding
the resulting mixture, and processing the mixture of granules, after adding
suitable auxiliaries if
desired, to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for
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example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically
acceptable polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be
added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a
salt thereof such as
sodium alginate.
Dragee cores arc provided with suitable coatings. For this purpose,
concentrated sugar
solutions may be used which may optionally contain gum arabic, talc, polyvinyl
pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and
suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for
identification or to characterize different combinations of active compound
doses.
Pharmaceutical compositions which can be used orally, include push-fit
capsules made of
gelatin as well as soft, sealed capsules made of gelatin and a plasticizer,
such as glycerol or sorbitol.
The push-fit capsules may contain the active ingredients in admixture with
filler such as lactose,
binders such as starches, lubricants such as talc or magnesium stearate and,
optionally, stabilizers.
In soft capsules, the active ingredients may be dissolved or suspended in
suitable liquids, such as
fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All
formulations for oral administration should be in dosages suitable for the
chosen route of
administration.
For buccal administration, the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
For administration by nasal inhalation, the active ingredients for use
according to some
embodiments of the invention are conveniently delivered in the form of an
aerosol spray
presentation from a pressurized pack or a nebulizer with the use of a suitable
propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or
carbon dioxide. In
the case of a pressurized aerosol, the dosage unit may be determined by
providing a valve to deliver
a metered amount. Capsules and cartridges of, e.g., gelatin for use in a
dispenser may be formulated
containing a powder mix of the compound and a suitable powder base such as
lactose or starch.
The pharmaceutical composition described herein may be formulated for
parenteral
administration, e.g., by bolus injection or continuous infusion. Formulations
for injection may be
presented in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an
added preservative. The compositions may be suspensions, solutions or
emulsions in oily or
aqueous vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or
dispersing agents.
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Pharmaceutical compositions for parenteral administration include aqueous
solutions of the
active preparation in water-soluble form. Additionally, suspensions of the
active ingredients may
be prepared as appropriate oily or water based injection suspensions. Suitable
lipophilic solvents
or vehicles include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate,
triglycerides or liposomes. Aqueous injection suspensions may contain
substances, which increase
the viscosity of the suspension, such as sodium carboxymethyl cellulose,
sorbitol or dextran.
Optionally, the suspension may also contain suitable stabilizers or agents
which increase the
s ol ubi 1 i ty of the active ingredients to all ow for the preparation of
highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution
with a suitable
vehicle, e.g., sterile, pyrogen-free water based solution, before use.
The pharmaceutical composition of some embodiments of the invention may also
be
formulated in rectal compositions such as suppositories or retention enemas,
using, e.g.,
conventional suppository bases such as cocoa butter or other glycerides.
Alternative embodiments include depots providing sustained release or
prolonged duration
of activity of the active ingredient in the subject, as are well known in the
art.
Pharmaceutical compositions suitable for use in context of some embodiments of
the
invention include compositions wherein the active ingredients are contained in
an amount effective
to achieve the intended purpose. More specifically, a therapeutically
effective amount means an
amount of active ingredients effective to prevent, alleviate or ameliorate
symptoms of a disorder
(e.g., cancer) or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the
capability of those
skilled in the art, especially in light of the detailed disclosure provided
herein.
For any preparation used in the methods of the invention, the therapeutically
effective
amount or dose can be estimated initially from in vitro and cell culture
assays. For example, a dose
can be formulated in animal models to achieve a desired concentration or
titer. Such information
can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein
can be
determined by standard pharmaceutical procedures in vitro, in cell cultures or
experimental
animals. The data obtained from these in vitro and cell culture assays and
animal studies can be
used in formulating a range of dosage for use in human. The dosage may vary
depending upon the
dosage form employed and the route of administration utilized. The exact
formulation, route of
administration and dosage can be chosen by the individual physician in view of
the patient's
condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1).
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Dosage amount and interval may be adjusted individually to provide levels of
the active
ingredient are sufficient to induce or suppress the biological effect (minimal
effective
concentration, MEC). The MEC will vary for each preparation, but can be
estimated from in vitro
data. Dosages necessary to achieve the MEC will depend on individual
characteristics and route
5 of administration. Detection assays can be used to determine plasma
concentrations.
Depending on the severity and responsiveness of the condition to be treated,
dosing can be
of a single or a plurality of administrations, with course of treatment
lasting from several days to
several weeks or until cure is effected or diminution of the disease state is
achieved.
The amount of a composition to be administered will, of course, be dependent
on the subject
10 being treated, the severity of the affliction, the manner of
administration, the judgment of the
prescribing physician, etc.
Compositions of some embodiments of the invention may, if desired, be
presented in a pack
or dispenser device, such as an FDA approved kit, which may contain one or
more unit dosage
forms containing the active ingredient. The pack may, for example, comprise
metal or plastic foil,
15 such as a blister pack. The pack or dispenser device may be accompanied
by instructions for
administration. The pack or dispenser may also be accommodated by a notice
associated with the
container in a form prescribed by a governmental agency regulating the
manufacture, use or sale
of pharmaceuticals, which notice is reflective of approval by the agency of
the form of the
compositions or human or veterinary administration. Such notice, for example,
may be of labeling
20 approved by the U.S. Food and Drug Administration for prescription drugs
or of an approved
product insert. Compositions comprising a preparation of the invention
formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an appropriate
container, and labeled for
treatment of an indicated condition, as is further detailed above.
According to another aspect of the present invention there is provided an
article of
25 manufacture comprising a packaging material packaging the T cell
disclosed herein; and a
therapeutic composition capable of binding a pathological cell and the TCR
complex.
According to specific embodiments, the article of manufacture is identified
for the
treatment of a disease associated with a pathologic cell (e.g. cancer).
According to specific embodiments, the T cells and the therapeutic composition
are
30 packaged in separate containers.
According to specific embodiments, the T cells and the therapeutic composition
are
packaged in a co-formulation.
As used herein the term "about" refers to 10 %
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The terms "comprises", "comprising", "includes", "including", "having" and
their
conjugates mean "including but not limited to".
The term "consisting of' means "including and limited to".
The term "consisting essentially of' means that the composition, method or
structure may
include additional ingredients, steps and/or parts, but only if the additional
ingredients, steps
and/or parts do not materially alter the basic and novel characteristics of
the claimed composition,
method or structure.
As used herein, the singular form "a", "an" and "the" include plural
references unless the
context clearly dictates otherwise. For example, the term "a compound" or "at
least one
compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be
presented in a
range format. It should be understood that the description in range format is
merely for
convenience and brevity and should not be construed as an inflexible
limitation on the scope of
the invention. Accordingly, the description of a range should be considered to
have specifically
disclosed all the possible subranges as well as individual numerical values
within that range. For
example, description of a range such as from 1 to 6 should be considered to
have specifically
disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to
4, from 2 to 6, from 3
to 6 etc., as well as individual numbers within that range, for example, 1, 2,
3, 4, 5, and 6. This
applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any
cited numeral
(fractional or integral) within the indicated range. The phrases
"ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges from" a first
indicate number
"to" a second indicate number are used herein interchangeably and are meant to
include the first
and second indicated numbers and all the fractional and integral numerals
therebetween.
As used herein the term "method" refers to manners, means, techniques and
procedures for
accomplishing a given task including, but not limited to, those manners,
means, techniques and
procedures either known to, or readily developed from known manners, means,
techniques and
procedures by practitioners of the chemical, pharmacological, biological,
biochemical and medical
arts.
When reference is made to particular sequence listings, such reference is to
he understood
to also encompass sequences that substantially correspond to its complementary
sequence as
including minor sequence variations, resulting from, e.g., sequencing errors,
cloning errors, or
other alterations resulting in base substitution, base deletion or base
addition, provided that the
frequency of such variations is less than 1 in 50 nucleotides, alternatively,
less than 1 in 100
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nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively,
less than 1 in 500
nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively,
less than 1 in 5,000
nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for
clarity, described in
the context of separate embodiments, may also be provided in combination in a
single embodiment.
Conversely, various features of the invention, which are, for brevity,
described in the context of a
single embodiment, may also be provided separately or in any suitable
subcombination or as
suitable in any other described embodiment of the invention. Certain features
described in the
context of various embodiments are not to be considered essential features of
those embodiments,
unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated
hereinabove and
as claimed in the claims section below find experimental support in the
following examples.
EXAMPLES
Reference is now made to the following examples, which together with the above
descriptions
illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized
in the present
invention include molecular, biochemical, microbiological and recombinant DNA
techniques.
Such techniques are thoroughly explained in the literature. See, for example,
"Molecular Cloning:
A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular
Biology"
Volumes I-BI Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular
Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A
Practical Guide to
Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al.,
"Recombinant DNA",
Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory
Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York
(1998);
methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;
5,192,659 and
5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-1,11 Cellis, J.
E., ed. (1994);
"Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-
Liss, N. Y. (1994),
Third Edition; "Current Protocols in Immunology" Volumes I-BI Coligan J. E.,
ed. (1994); Stites
et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange,
Norwalk, CT
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology",
W. H. Freeman
and Co., New York (1980); available immunoassays are extensively described in
the patent and
scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3.901,654; 3,935,074; 3,984,533; 3,996.345;
4,034,074;
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4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis"
Gait, M. J., ed.
(1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription
and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell
Culture" Freshney,
R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to
Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317,
Academic
Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press,
San Diego, CA
(1990); Marshak et al., "Strategies for Protein Purification and
Characterization - A Laboratory
Course Manual" CSHL Press (1996); all of which are incorporated by reference
as if fully set forth
herein. Other general references are provided throughout this document. The
procedures therein
are believed to be well known in the art and are provided for the convenience
of the reader. All
the information contained therein is incorporated herein by reference.
MATERIALS AND METHODS
Cell culture and Reagents - K562, Raji and 293T cell lines (JCRB) were
cultured as
recommended by the supplier. All cell lines were authenticated by short tandem
repeat (STR)
profiling using PowerPlex16 HS kit (Promega). Cell number and viability were
estimated using
hemocytometer and trypan blue exclusion assay, respectively. Research grade of
Blinatumomab
and MT110 were purchased from Oak BioSciences, Inc (CA, USA).
Generation of endogenous TCR negative T cells - Human peripheral blood
mononuclear
cells (PBMCs) cells were purified from peripheral blood samples of healthy
donors, using Ficoll-
Hypaque density gradient centrifugation. Extracted PBMCs were activated and
expanded with
OKT3 (Biolegend, Cat. No 317301), in 24-well suspension plates in 4Ce110 Nutri-
T Media
(Sartorius, 05-11F2001-1K) supplemented with 500 U / ml of IL-2 (Peprotech,
200-02) and
antibiotics. 72 hours following activation cells were electroporated using
AMAXA Nucleofector
(Lonza) with SpCas9 RNPs using Alt-R CRISPR-Cas9 System and sgRNA that target
the human
constant alpha region (SEQ ID NO: 1) or the human constant beta region (SEQ ID
NO: 45) (IDT,
Coralville). Electroporated cells were cultured in 4Ce110 Nutri-T Media
supplemented with 500
U / ml of IL-2 and antibiotics. Three days following electroporation, most of
the cells were
negative for the constant alpha or beta chain (> 85 %) determined by anti-CD3
flow antibody (PE-
Cy7 OKT3 Biolegend, Cat No. 317333). The negative cells were further enriched
by magnetic
anti-CD3 beads (Miltenyi Biotec, Cat No. 130-050-101), following
manufacturers' instructions,
to reach 95%-99.9% CD3-/TCR- cells (Figure 3A). For knocking out both alpha
and beta chains,
the cells were firth electroporated with gRNA targeting the alpha chain (SEQ
ID NO: 1),
negatively selected by magnetic beads and then were further electroporated
with sgRNA targeting
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the beta region (SEQ ID NO: 45), followed by magnetic beads purification of
the CD3-/TCR- T
cells.
DNA constructs and cloning - An RTV-020 retroviral vector (Cell Biolabs Inc.
San
Diego, CA) was used. For each construct, the amino acid sequence was optimized
(IDT,
Coralville), each sequence was designed to express a polypeptide encoding a
different TCR alpha
and/or beta polypeptides, V5-furin-P2A sequence (S Yang, et al. 2008) and EGFP
or truncated
NGFR (ANGFR) as indicated. Schematic representations of the designed
constructs are shown in
Figures 2A-B, amino acid sequences are provided in SEQ ID NOs: 2-3, 26-32, 46,
48-52, 57, 59,
61-69, 109, 123, 125 and 127; nucleic acid sequences are provided in SEQ ID
NOs: 33-41, 53-56,
58, 60, 70-80, 110, 124, 126 and 128). The final sequences were ordered as
gBlocks (IDT,
Coralville). gBlocks were amplified using proper primers and High fidelity
PrimeSTAR GXL
DNA Polymerase (Cat No. R050B, TAKARA) then digested with BamHI-HF and XhoI
(NEB)
ligated into the retroviral vector. When indicated, 3 mutations were
introduced in the
transmembrane domain of the alpha chain: S116L, G119V and F120L (marked as
LVL). When
indicated, cysteines were introduced in both the alpha and beta chains (Cys):
T48C in the alpha
chain and S57C in the beta chain, L12C in the alpha chain and S17C in the beta
chain, Y43C in
the alpha chain and L63C in the beta chain, S61C in the alpha chain and R79C
in the beta chain,
L12C in the alpha chain and F14C in the beta chain, V22C in the alpha chain
and F14C in the beta
chain, YlOC in the alpha chain and Sl7C in the beta chain, T45C in the alpha
chain and D59C in
the beta chain, L50C in the alpha chain and S57C in the beta chain, S61C in
the alpha chain and
S57C in the beta chain, T45C in the alpha chain and S77C in the beta chain,
S15C in the alpha
chain and V13C in the beta chain, or S15C in the alpha chain and E15C in the
beta chain. When
indicated (marked as mm), several mutations were introduced in the
extracellular domain of the
alpha and beta chains, as follows: alpha chain mutations P91S, E92D, S93V,
S94P; beta chain
mutations: E18K, S22A, F1331, E/V136A, Q139H. When indicated (marked as Des),
several
mutations were introduced in the alpha and beta chains, as follows: alpha
chain mutations S21F,
T32I, A72T and beta chain mutations E18K, H23R, D39P, S54D. Sanger sequencing
was used to
validate cloned vectors. In the same manner, Ba9 (SEQ ID NO: 83-84) and a6
(SEQ ID NO:81-
82) constructs disclosed by International Patent Application Publication NO.
W02020138371
were designed and cloned.
Virus preparation and transduction procedure - Viral particles were generated
by
transient transfection of 293T cells with the contracted retroviral vector
using CMV-gagPol and
CMV-VSVG (Cell Biolabs, Inc.). CRISPR-edited TCR negative T cells were seeded
on tissue
culture plates coated with Retronectin (TaKaRa Bio) and the concentrated viral
particles were
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added. Cells were incubated with the viral preparations for 7 hours, and then
the medium was
replaced with 4Ce110 Nutri-T Media supplemented with 500 U / ml of IL-2 and
antibiotics.
Flow cytometry analysis ¨ Cells were stained with OKT3 Anti-CD3 ¨APC
(Biolegend),
following manufacturers' instructions. Results were obtained using GalliosTM
and Cytoflex Flow
5
Cytometers (Beckman Coulter, Inc.). Flow cytometry results were analyzed
using FlowJo v10
software.
In vitro anti-tumor effect ¨ Endogenous TCR negative T cells were infected
with BluT
comprising a T48C mutation and the LVL mutations and a truncated beta chain
comprising a S 57C
mutation (SEQ ID NO: 125-126); and selected with magnetic beads using the
ANGFR marker.
10
Following, cells were incubated for 24 hours at 37 'V with Raji-F.Luc CD19+
lymphoma cells
(Cat.1F050046 JCRB) at various E : T ratios with or without 500 lug / ml
Blinatumomab (a CD19
BiTE) and firefly luciferase activity was measured by GloMax0 Discover
Microplate Reader to
determine cytotcodcity. Anti-CD19 CAR-T cells were produced by infecting
endogenous TCR
negative T cells with anti-CD19(FMC63)-CD28z (SEQ ID NO: 127-128) and
selection with
15
magnetic beads using the ANGFR marker ; and un-modified CD3 positive TCR
positive T cells
were used as positive controls.
In vivo anti-tumor effect ¨2x106 Raji-F.Luc were injected subcutaneously to
NSG mice
with (n = 34) or without (n = 8) 107 T cells (endogenous TCR negative T cells
infected with a
vector encoding a truncated alpha chain comprising a T48C mutation and the LVL
mutations and
20
a truncated beta chain comprising a S57C (SEQ ID NO: 125-126) and selected
with magnetic
beads using ANGFR marker (n = 10); endogenous TCR negative anti-CD19 CAR-T
cells infected
with anti-CD19(FMC63)-CD28z and selected with magnetic beads using the ANGFR
marker (n =
8); or un-modified CD3 positive TCR positive T cells (n = 16). 24 hours
following injection, the
indicated groups were treated i.v. with 5 tg / mouse Blinatomumab for 5
consecutive days.
25
Bioluminescence imaging (1V1S0 Lumina X5, PerkElmer) was performed once a
week up to day
to track tumor growth.
EXAMPLE 1
GENERATION OF A TRUNCATED T CELL RECEPTOR (TCR)
30
The present inventors have devised a novel engineered TCR that is devoid of
the variable
regions of the TCR alpha and beta chains. This TCR, referred to herein as
"blunt truncated TCR
(BluT)", is presented as part of the TCR complex on the cell surface (Figure
1A) and can serve as
a TCR that does not recognize any antigen through the MHC machinery, thus
preventing the
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56
induction of GvHD even in cases of mismatch pairing with the endogenous TCR
alpha or beta
chains (Figure 1B).
To this end, several constructs encoding human TCR alpha and/or beta chains
devoid of
their variable regions were designed (Figures 2A-B, amino acid sequences are
provided in SEQ
ID NOs: 2-3 and 26-32, 46-52, 57, 59, 61-69, 81, 83, 109, 123, 125 and 127 ;
nucleic acid
sequences are provided in SEQ ID NOs: 33-41. 53-56, 58, 60, 70, -80, 82, 84,
124, 126 and 128),
packed into retroviruses and used to infect TCR negative T cells (formed by
CRTSPR targeting of
the TCR alpha chain) (Figures 3A-B). EGFP was used as a marker of stable
infection. As shown
in Figures 3B and 4A and Table 1 hereinbelow, introducing a vector encoding
only a truncated
alpha chain or only a truncated beta chain did not result in presentation of a
TCR complex on the
cell surface (manifested by no expression of CD3 on the surface). In addition,
introducing a vector
encoding a truncated alpha and beta (either TRBC1 or TRBC2) chains did not
result in presentation
of a TCR complex on the cell surface.
To solve this problem the present inventors searched for modifications that
will stabilize
the truncated TCR (Figures 3B-8B and Table 1 hereinbelow).
Introducing a vector encoding a truncated alpha chain comprising the
stabilizing mutations
S116L, G119V and F120L (marked as LVL) and a vector encoding a truncated beta
chain; or a
vector encoding truncated alpha and beta chains comprising computationally
designed stabilizing
mutations, namely S21F, T32I, A72T in the alpha chain and E18K, H23R, D39P.
S54D in the beta
chain (marked as Des), did not result in presentation of a TCR complex on the
cell surface (Figures
4 and 7). Moreover, trying to express in primary T cells not expressing
endogenous alpha chain,
the best ranked truncated alpha chain disclosed by International Patent
Application Publication
NO. W02020138371 alone or with full constant beta chain, did not lead to CD3
re-expression
(Figure 8B).
However, introducing a vector encoding a truncated alpha chain comprising a
T48C
mutation and a truncated beta chain comprising a 557C leading to formation of
an additional
disulfide bond between the alpha and beta chains resulted in presentation of a
TCR complex on
the cell surface (manifested by re-expression of CD3 on the surface) (Figures
3B and 4). Further,
addition of the LVL mutations to the T48 / S57 cysteine mutations improved CD3
re-expression
on the cell surface (Figure 4). Importantly, introducing mutations leading to
an additional di sulfide
bond between the alpha and beta chains in other applicable cysteine sites (SEQ
ID NO: 85-108;
see e.g. Boulter et al., 2003), did not enable CD3 re-expression (Figure 6).
Further, it was found
that removal of 11 amino acids or more from the N terminal of the constant
domain of the alpha
chain destabilize the complex and prevent CD3 expression (Figure 8A).
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In addition, introducing a vector encoding a truncated alpha and beta chains
comprising
minimal murine amino acid substitutions (marked as mm), namely: P91S, E92D,
S93V, S94P in
the alpha chain and E18K, S22A, F1331, E/V136A, Q139H in the beta chain, also
resulted in
presentation of a TCR complex on the cell surface (manifested by re-expression
of CD3 on the
surface) (Figure 7). Same as with the cysteine mutations, adding the LVL
modifications to the
mm modifications further enhanced CD3 re-expression.
To show that these results are not exclusively valid when knocking out the
endogenous
alpha chain to prevent cell surface expression of the endogenous TCR, SpCas9
RNP was used to
target the endogenous beta chain (Figure 5). In the same manner, while
introducing a vector
encoding truncated alpha and beta chains did not result in presentation of a
TCR complex on the
cell surface; introducing a vector encoding a truncated alpha chain comprising
a T48C mutation
and a truncated beta chain comprising a S57C mutation resulted in presentation
of a TCR complex
on the cell surface (manifested by re-expression of CD3 on the surface).
Knocking out both alpha
and beta endogenous chains had no further effect.
Taken together, in order to express on the surface of T cells a TCR complex
comprising
human TCR alpha and beta chains devoid of their variable domains,
modifications to the human
TCR wild type sequences must be introduced. The following possible and
sufficient modifications
were identified: replacing T48 of the alpha chain and S57 of the beta chain
with cysteine residues;
and/or replacing several residues in both chains with their rnurine
counterparts. Further
introducing LVL mutations in the alpha chain improves cell surface expression.
Table 1:
SEQ Construct name (see
CD3 MFI Ration
ID Figure 2A for schematic % of CD3+ % of CD3+ in
%EGFP
between
NO: representations of the in EGFP- EGFP+
EGFP+/EGFP-
constructs)
Non infected control 0 95 0
0
26/34 EGFP-P2A-TRAC(Cys)-
4.53 40.5 7.537
P 2A-TRBC 1 (Cy s)
27/35 EGFP-P2A-TRAC(Cys)-
FurinV5P2A- 27 4.3 48
9.85
TRBC 1 (Cys)
2/33 EGFP-P2A-
TRAC(Cys,LVL)-P2A- 31 4.45 48.4
11.29
TRBC1(Cys)
28/36 EGFP-P2A-
TRAC(Cys,LVL)-
18 3.9 68
34.5
FurinV5P2A-
TRBC 1 (Cys)
29/37 EGFP-P2A-mmTRAC-
20 4.25 45
20.8
FurinV5P2A-mmTRBC1
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30/38 EGFP -P2A-
mmT RAC (Cy s)-
15 3.33 55.733
27.05
FurinV5P2A-
mmTRBC1(Cys)
31/39 EGFP -P 2A-
mmT RAC (LVL)- 27 4.73 69.63
31.033
FurinV5P2A-mmTRBC1
32/40 EGFP-P2A-
mmTRAC(Cys,LVL)-
24 3.61 67.9
35.25
FurinV5P2A-
mmTRBC1(Cys)
EXAMPLE 2
T CELLS EXPRESSING BluT TCR IN COMBINATION WITH A BI-SPECIFIC T
CELL ENGAGER INDUCE EFFECTIVE IN-VITRO TUMOR CELL LYSIS
As the therapeutic strategy of some embodiments is based on infusing to a
subject T cells
expressing the BluT along with a bi-specific antibody binding the TCR complex
on one hand (e.g.
CD3) and a cancer antigen on the other hand, the cytotoxic activity of the
transduced T cells can
be evaluated in-vitro on e.g. CD19+ Raji cells and/or EGER+ K562 cells in
combination with the
anti-CD19 bispecific T-cell engager (BiTE)- Blinatumomab and/or the anti EPCAM
BiTE-
MT110. T cell activation can be determined by e.g. expression of CD107 and
secretion of
cytoki nes such as IFNy, TNFa, IL-2. Cytotoxic activity can be determined by
e.g. change in RLU,
reduction in CD19 or EGFR percentage in comparison with non-treated controls.
For positive
selection of transduced T cells with BluT, ANGER may be used as a selection
marker (instead of
the EGFP described in Figures 2A-B and Example 1 hereinabove).
To this end, endogenous TCR negative T cells expressing a truncated alpha
chain
comprising a T48C mutation and the LVL mutations and a truncated beta chain
comprising a S57C
were incubated for 24 hours with Raji-F.Luc CD19 positive lymphoma line at
various E : T ratios
with or without Blinatumomab (a CD19 BiTE) and firefly luciferase activity was
measured to
determine cytotoxicity (Figure 9). Endogenous TCR negative anti-CD19 CAR-T
cells and un-
modified CD3 positive TCR positive T cells were used as positive controls.
Unlike unmodified T cells that contain full endogenous TCR (which can
recognize tumor
cell through 1V1HC-TCR machinery and as a result have cytotoxic activity
against the target cells
without adding BiTE), the T cells comprising the modified truncated TCR have
an anti-tumor
activity only in the presence of an anti-CD3 mediator. Further, the T cells
comprising the modified
truncated TCR were significantly more cytotoxic compared to the unmodified T
cells or the anti-
CD19 CAR-T cells.
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EXAMPLE 3
T CELLS EXPRESSING BluT TCR IN COMBINATION WITH A BI-SPECIFIC T
CELL ENGAGER INDUCE EFFECTIVE IN-VIVO ANTI-TUMOR EFFECR
As the therapeutic strategy of some embodiments is based on infusing to a
subject T cells
expressing the BluT along with a bi-specific antibody binding the TCR complex
on one hand (e.g.
CD3) and a cancer antigen on the other hand, the cytotoxic activity of
endogenous TCR negative
T cells expressing a truncated alpha chain comprising a T48C mutation and the
LVL mutations and
a truncated beta chain comprising a S57C was evaluated in-vivo in mice
transplanted with CD19+
Raji cells in combination with the anti-CD19 BiTE Blinaturnomab. Endogenous
TCR negative
anti-CD19 CAR-T cells and un-modified CD3 positive TCR positive T cells were
used as positive
controls.
As shown in Figure 10, the T cells comprising the modified truncated TCR have
an anti-
tumor activity only in the presence of the anti-CD3 mediator.
Although the invention has been described in conjunction with specific
embodiments
thereof, it is evident that many alternatives, modifications and variations
will be apparent to those
skilled in the art. Accordingly, it is intended to embrace all such
alternatives, modifications and
variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the applicant(s) that all publications, patents and patent
applications
referred to in this specification are to be incorporated in their entirety by
reference into the
specification, as if each individual publication, patent or patent application
was specifically and
individually noted when referenced that it is to be incorporated herein by
reference. In addition,
citation or identification of any reference in this application shall not be
construed as an admission
that such reference is available as prior art to the present invention. To the
extent that section
headings are used, they should not be construed as necessarily limiting. In
addition, any priority
document(s) of this application is/are hereby incorporated herein by reference
in its/their entirety.
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